Method for determining the substrate specificity of an enzymatic activity and a device therefor

ABSTRACT

The invention relates to a method for determining the substrate specificity of an enzymatic activity comprising the following steps: providing an assembly comprising a plurality of amino acid sequences on a planar surface of a support material, whereby the amino acids are directionally immobilised; contacting and/or incubating of an enzymatic activity with the assembly; and detection of a reaction between one of the amino acid sequences that are immobilised on the assembly and the enzymatic activity. According to the invention, during the reaction of the enzymatic activity with the assembly, a change in the molecular weight of at least one of the amino acid sequences takes place.

[0001] The present invention relates to assemblies of a plurality ofamino acid sequences on a surface, supports and support assembliescomprising these, a method for producing such an assembly, a method fordetermining the substrate specificity of an enzymatic activity, use ofthe method for determining the pattern of the enzymatic activity of asample.

[0002] With the increasing availability of sequence information from thevarious genome projects, the assembly of nucleic acid fragments having ahigh density on a support material, so-called chips or biochips, hasacquired major importance whose full potential, however, has only beenable to be utilised with the availability of newer synthesis techniquesand miniaturisation, and has resulted in a plurality of applications. Inaddition to nuclei acids, natural substances or libraries thereof butalso assemblies of oligopeptides and proteins have been applied to suchchips. Cellulose, glass, nitrocellulose, PFTE membranes and special agarhave been used as support materials for these assemblies.

[0003] With the increasing importance of proteomics and itsbiotechnological application, peptides and proteins have become thefocus of interest. In general, it is proteins and mostly their enzymaticactivities which make possible almost all biochemical reactions insideand outside the cell. The use of assemblies of nucleic acids with whicheither the messenger RNA (mRNA), which was generated by the genesspecifically active in the cell, or DNA copies of this mRNA aredetected, is certainly of major importance but the informationobtainable therewith is for many reasons not sufficient forunderstanding the processes involved in both intracellular andextracellular processes and for use to be made thereof in variousbiotechnological applications. One reason for this is that the quantityof mRNA in a cell frequently does not correlate with the correspondingamount of protein produced in the cell. In addition, proteins onceproduced can be considerably influenced in their enzymatic activity (andthus in their biological function) by slight chemical modifications inthe cell (post-translational modifications). There is thus a need tocarry out a parallel analysis of the enzymatic activity of as manyproteins as possible, especially enzymes. Such an approach allows, amongother things, the substrate specificity of a defined enzyme to bedetermined rapidly which is again an important requirement for thedesign of knowledge-based inhibitors, or for the selective testing ofpharmaceuticals or pharmaceutical candidates, especially as part of theprediction of side effects.

[0004] In the prior art assemblies of peptides or proteins wereimmobilised on various surfaces such as glass (J. Robles, M. Beltran, V.Marchan Y. Perez, I. Travesset, E. Pedroso, A. Grandas; 1999, Towardsnucleotides containing any trifunctional amino acid, Tetrahedron, 55,13251-13264), cellulose (D. R. Englebretsen, D. R. K. Harding; 1994,High yield, directed immobilization of a peptide-ligand onto a beadedcellulose support, Pept. Res. 7, 322-326), nitrocellulose (S. J.Hawthorne, M. Pagano, P. Harriott, D. W. Halton, B. Walker; 1998, Thesynthesis and utilisation of 2,4-dinitrophenyl-labeled irreversiblepeptidyl diazomethyl ketone inhibitors, Anal. Biochem., 261, 131-138),PTFE membranes (T. G. Vargo, E. J. Bekos, Y. S. Kim, J. P. Ranieri, R.Bellamkonda, P. Aebischer, D. E. Margevich, P. M. Thompson, F. V.Bright, J. A. Gardella; 1995, Synthesis and characterization offluoropolymeric substrata with immobilized minimal peptide sequences forcell adhesion studies. 1., J. Biomed. Mat. Res. 29, 767-778), titaniumoxide (S. J. Xiao, M. Textor, N. D. Spencer, M. Wieland, B. Keller, H.Sigrist; 1997, Immobilization of the cell-adhesive peptideARG-GLY-ASP-CYS(RGDC) on titanium surfaces by covalent chemicalattachment, J. Materials Science-Materials in Medicine, 8, 867-872),silicon oxide (T. Koyano, M. Saito, Y. Miyamoto, K. Kaifu, M. Kato;1996, Development of a technique for microimmobilization of proteins onsilicon wafers by a streptavidin-biotin reaction, Biotech. Progress.,12, 141-144) or gold (B. T. Houseman, M. Meksich; 1998, Efficientsolid-phase synthesis of peptide-substituted alkanethiols for thepreparation of substrates that support the adhesion of cells, J. Org.Chem. 63, 7552-7555) or the stepwise synthesis of peptides was carriedout directly on a corresponding glass surface (S. P. A. Fodor, J. L.Read, M. C. Pirrung, L. Stryer, A. T. Lu, D. Solas; 1991,Light-directed, spatially addressable parallel chemical synthesis,Science, 251, J. P. Pellois, W. Wang, X. L. Gao; 2000, Peptide synthesisbased on t-Boc chemistry and solution photogenerated acids, J. Comb.Chem. 2, 355-360) or on cellulose (R. Frank, 1992, Spot synthesis: aneasy technique for the positionally addressable, parallel chemicalsynthesis on a membrane support, Tetrahedron, 48, 9217-9232; A Kramerand J. Schneider-Mergener, Methods in Molecular Biology, vol 87:Combinatorial Peptide Library Protocols, p. 25-39, edited by: S.Cabilly; Humana Press Inc., Totowa, N.J.; Töpert, F., Oires, C.,Landgraf, C., Oschkinat, H. and Schneider-Mergener, J., 2001, Synthesisof an array comprising 837 variants of the hYAP WW protein domain,Angew. Chem. Int. Ed., 40, 897-900) or on polypropylene (M. Stankova, S.Wade, K. S. Lam, M. Lebl; 1994, Synthesis of combinatorial librarieswith only one representation of each structure, Pept. Res. 7, 292-298,F. Rasoul, F. Ercole, Y. Pham, C. T. Bui, Z. M. Wu, S. N. James, R. W.Trainor, G. Wickham, N.J. Maeji; 2000, Grafted supports in solid-phasesynthesis, Biopolymers, 55, 207-216, H. Wenschuh, R. Volkmer-Engert, M.Schmidt, M. Schulz, J. Schneider-Mergener, U. Reineke; 2000, Coherentmembrane supports for parallel microsynthesis and screening of bioactivepeptides, Biopolymers, 55, 188-206) or on chitin (W. Neugebauer, R. E.Williams, J. R. Barbier, R. Brzezinski, G. Willick; 1996, Peptidesynthesis on chitin, Int. J. Pept. Prot. Res. 47, 269-275) or onSepharose (W. Tegge, R. Frank, 1997, Peptide synthesis on Sepharosebeads, J. Peptides Res., 49, 355-362, R. Gast, J. Glokler, M. Hoxter, M.Kiess, R. Frank, W. Tegge; 1999. Method for determining protein kinasesubstrate specificities by the phosphorylation of peptide libraries onbeads, phosphate-specific staining, automated sorting, and sequencing,Anal. Biochem., 276, 227-241).

[0005] The object of the present invention is thus to provide a meansfor testing substrate specificities of enzymatic activities which on theone hand is suitable for use in a system with high throughput and on theother hand, can be carried out with extremely small quantities ofenzymatic activity or sample volume. It is especially an object that themeans has an improved signal-to-noise ratio compared with the meansaccording to the prior art, especially the peptide and proteinassemblies described therein and there described as “arrays”.

[0006] Another object of the present invention is to provide a methodfor producing such means and a method for determining the substratespecificity of an enzymatic activity and a method for determining theselectivity of an active substance.

[0007] This object is solved according to the invention by a method fordetermining the substrate specificity of an enzymatic activitycomprising the following steps:

[0008] Preparation of an assembly comprising a plurality of amino acidsequences on a planar surface of a support material wherein the aminoacid sequences are directionally immobilised,

[0009] Contacting and/or incubating of an enzymatic activity with theassembly, and

[0010] Detection of a reaction between one of the amino acid sequencesimmobilised on the assembly and the enzymatic activity,

[0011] wherein it is provided that during the reaction of the enzymaticactivity with the assembly, a change in the molecular weight of at leastone of the amino acid sequences takes place.

[0012] In one embodiment it is provided that the reaction is detected onor using the amino acid sequence immobilised on the surface of thesupport material.

[0013] In another embodiment it is provided that the change in themolecular weight takes place by formation or cleaving of a covalent bondon one of the amino acid sequences, preferably on that amino acidsequence which reacts with the enzymatic activity.

[0014] In yet another embodiment it is provided that the reaction isdetected by detecting the change in the molecular weight.

[0015] Finally in one embodiment it is provided that the reaction isdetected by a detection method selected from the group comprisingautoradiography, plasmon resonance spectroscopy and fluorescencespectroscopy.

[0016] In one embodiment it is provided that at least one of the aminoacid sequences is a substrate for an enzymatic activity.

[0017] In another embodiment it is provided that the assembly of aminoacid sequences for at least two different enzymatic activities has atleast one substrate each.

[0018] In a preferred embodiment it is provided that the enzymaticactivity is selected from the group comprising kinases,sulphotransferases, glycosyl transferases, acetyl transferases, farnesyltransferases, palmytyl transferases, phosphatases, sulphatases,esterases, lipases, acetylases and proteases.

[0019] In another embodiment it is provided that the detection of areaction between the amino acid sequences immobilised on the assemblyand the enzymatic activity is repeated many times, preferably afterintervals of time.

[0020] In yet another embodiment it is provided that the enzymaticactivity is determined in a sample and the sample is preferably selectedfrom the group comprising urine, liquor, sputum, stool, lymph fluid,cell lysates, tissue lysates, organ lysates, extracts, raw extracts,purified preparations and unpurified preparations.

[0021] In one embodiment it is provided that the surface is a non-poroussurface.

[0022] In another embodiment it is provided that the support material isglass.

[0023] In yet another embodiment it is provided that the amino acidsequence is immobilised via a sulphur-comprising group on the surface.

[0024] In a second aspect the object is solved by an assembly of aplurality of amino acid sequences on a surface, preferably on thesurface of a solid-phase support, wherein the amino acid sequences aredirectionally immobilised on the planar surface of a support material,wherein at least one of the amino acid sequences is a substrate for anenzymatic activity, wherein a change in the molecular weight takes placeon the substrate as a result of the enzymatic activity.

[0025] In one embodiment it is provided that the change in the molecularweight takes place as a result of the formation or cleavage of acovalent bond on the substrate.

[0026] In another embodiment it is provided that the assembly of aminoacid sequences for at least two different enzymatic activities has atleast one substrate each.

[0027] In yet another embodiment it is provided that the planar surfaceis a non-porous surface.

[0028] In one embodiment it is provided that the support material isselected from the group comprising silicates, ceramic, glass, metals andorganic support materials.

[0029] In another embodiment it is provided that the amino acidsequences are selected from the group comprising peptides,oligopeptides, polypeptides and proteins as well as their respectivederivatives.

[0030] In yet another embodiment it is provided that each amino acidsequence or group of amino acid sequences has a defined arrangementrelative to another amino acid sequence or groups of amino acidsequences.

[0031] In another aspect the object according to the invention is solvedby a support comprising an assembly according to the invention.

[0032] In one embodiment it is provided that the support comprises abase support material.

[0033] In another embodiment it is provided that the assembly of aplurality of amino acid sequences is arranged on one or a plurality ofsurfaces of the support.

[0034] In another aspect the object is solved by a support assemblycomprising at least two supports according to the invention, whereinrespectively two supports are separated by a gap.

[0035] In one embodiment it is provided that at least one assembly on afirst support is facing at least one assembly on a second support.

[0036] In another embodiment it is provided that the gap has a width ofaround 0.01 mm to 10 mm, preferably around 0.1 mm to 2 mm, and morepreferably around 0.5 mm to 1 mm.

[0037] In yet another aspect the object is solved according to theinvention by the use of an assembly according to the invention and/or asupport according to the invention and/or a support assembly accordingto the invention in a method according to the invention.

[0038] The present invention is based on the surprising finding (seeFIG. 12A) that with an assembly of a plurality of amino acid sequences(see FIG. 12A, B₁-B₃) on a surface, wherein it is especially providedthat the amino acid sequences are directionally immobilised on thesurface and the surface is a planar surface, on bringing the assemblyinto contact with a sample containing a potential interaction partner(FIG. 12A, C) for one or a plurality of amino acid sequences containedin the assembly, very small quantities of the potential interactionpartner, expressed as international units/liquid volumes, can suffice todetect a binding event between one or a plurality of the amino acidsequences and the potential interaction partner.

[0039] The potential interaction partner is preferably an enzymaticactivity and the binding event is the formation of the complex ofenzymatically active protein and—potential—substrate required for acatalytic reaction. In other words, the assembly according to theinvention allows the signal-to-noise ratio to be improved by severalorders of magnitude compared with the assemblies according to the priorart, which is based on the special combination of the features of thedirectional immobilisation and the presence of a planar surface.

[0040] When porous surfaces are used, as is the case for example, whenusing cellulose or porous glass, a large quantity of material, in thepresent case of amino acid sequences per unit area, can be immobilised,which results in good signal intensities and large regions with aproportional measurement signal, but at the same time the availabilityof the large surface causes a non-specific interaction of the amino acidsequences with the support material which leads to higher backgroundsignals. Furthermore, such porous surfaces require substantially morematerial to develop the assembly or for coating a support materialcarrying the assembly, i.e., larger quantities of each of the variousamino acid sequences. Likewise as a result of the porous surface, moresample material is required for the actual analysis process. The samplematerial comprises such material that contains a possible interactionpartner for one or a plurality of amino acid sequences. However, thisincrease in sample material cannot be compensated in every case byproviding a larger sample volume, but rather it may be necessary toincrease the specific quantity of the potential interaction partner inthe sample which comes in contact or should come in contact with theassembly. This would necessitate purifying the sample material to beanalysed wherein however quite appreciable losses frequently occurduring such purification, so that the use of porous surfaces forassemblies of molecules on surfaces is not suitable for detectinginteraction partners whose concentration in a sample is comparativelylow. If the potential interaction partner comprises an enzymaticactivity (which herein generally includes enzymes and any catalyticallyactive molecules, for example, also catalytically active nucleic acids),under the influence of the purification or concentration of the samplematerial or the interaction partner, i.e., the specific enzymaticactivity, required when using assemblies according to the prior art, thesituation may arise that certain enzymatic activities cannot bedetermined. This imposes a considerable limitation on the use ofassemblies comprising amino acid sequences insofar as it is frequentlythose enzymatic activities which are not necessarily the predominantquantity in a sample, that are of central biological importance. Thus,with the assembly according to the invention for example, maceratedcells can be analysed without further treatment in the sense ofpurification and enzymatic interaction partners contained therein can bedetected with a low specific activity.

[0041] A further disadvantage of using porous surfaces is that capillaryforces unavoidably act there, preventing any miniaturisation as isespecially required for high-throughput systems. In other words, whenporous support systems are used, only a certain density of amino acidsequences can be achieved in an assembly. Currently, as a result of thephysico-chemical properties forming the basis of the porosity, thislimit in the case of cellulose is 100/cm².

[0042] On the other hand, however, the use of planar surfaces alone isagain not suitable for preparing an assembly to move forward into therange of signal intensities attainable with the assembly according tothe invention, especially the signal-to-noise ratios, since the loadingcapacity is frequently the limiting factor here. Attempts to avoid theselimitations by applying polyacrylamide gels having a defined pore widthto the planar, non-porous surface did not result in the desired successsince the said disadvantages of the porous membranes were subsequentlyreintroduced again here.

[0043] With the present invention a method is adopted for theconstruction of assemblies of a plurality of amino acid sequences on asurface, which not only concentrates on the surface aspect but also onthe specific type of immobilisation of the amino acid sequencescontained on the assembly, and thus accounts for their surprisingperformance. The planar surface merely requires a comparatively smallquantity of different amino acid sequences which in addition, as aresult of their directional immobilisation on the surface, presentoptimal interaction partners, especially substrates for enzymaticactivities so that despite the comparatively low loading capacity as aresult of the smooth, i.e., preferably non-porous surface, significantsignals are nevertheless achieved and likewise as a result of the planarsurface, no non-specific absorption occurs and therefore nodeterioration in the signal-to-noise ratio. As is shown by means of themodel calculation given in the examples, as a result of the combinationof these two features of the assembly according to the invention, thesignal-to-noise ratio is improved by a factor of 3000, as can be seenfrom FIGS. 3 and 4.

[0044] In the assembly according to the invention of a plurality ofamino acid sequences on a surface, the surface functions to a certainextent as a substrate on which the plurality of amino acid sequences isimmobilised. The immobilisation can take place such that it isaccomplished covalently. In addition to covalent immobilisation,however, other forms of immobilisation are also possible, especiallyadsorptive immobilisation or immobilisation via specific interactionsystems. Especially preferred for the immobilisation is covalentimmobilisation wherein a chemoselective binding of the amino acidsequences to the surface of the support material takes place. A numberof reactions known as such to the person skilled in the art can be usedhere (Lemieux, G. A. & Bertozzi, C. R., 1998, Chemoselective ligationreactions with proteins, oligosaccharides and cells, TIBTECH, 16,506-513, see FIG. 11 for this). With a view to the required directionalimmobilisation it should basically be ensured that under the respectiveinteraction conditions, substantially only one special compound isformed between the amino acid sequence and the surface (FIG. 12, LinkerA). The choice of the reactive group on the amino acid sequence sidewill thus depend substantially on the individual sequence. Alternativelyit is provided within the scope of the present invention that a terminalstructure standard to all the amino acid sequences is provided and thisterminal structure is made available for the specific reaction with thesurface, especially an activated surface (FIG. 12, Linker A). Typicallyduring the chemoselective reactions amino or carboxyl groups containedin the amino acid sequence are not adversely affected. Examples ofsuitable reactions are the formation of thioethers from halo-carbonicacids and thiols, which include the formation of thioethers fromhalocarbonic acids and thiols, thioethers from thiols and maleinimides,amide bonds from thioesters and 1,2-aminothiols, thioamide bonds fromdithioesters and 1,2-aminothiols, thiazolidines from aldehydes and1,2-aminothiols, oxazolidines from aldehydes/ketones and 1,2-aminoalcohols, imidazoles from aldehydes/ketones and 1,2-diamines (see alsoFIG. 11), thiazols from thioamides and alpha-halo-ketones, aminothiazolsfrom amino-oxy-compounds and alpha-isothiocyanato-ketones, oximes fromamino-oxy-compounds and aldehydes, oximes from amino-oxy-compounds andketones, hydrazones from hydrazines and aldehydes, hydrazones fromhydrazides and ketones. Moreover, the radicals R1-R5 shown in FIG. 11 orthe residues in the above-mentioned chemoselective reactions can bealkyl, alkenyl, alkynyl, cycloalkyl or aryl radicals or heterocycliccompounds, wherein alkyl stands for branched and unbranched C₁₋₂₀-alkyl,C₃₋₂₀-cycloalkyl, preferably for branched and unbranched-C₁₋₁₂ alkyl,C₃₋₁₂-cycloalkyl, and especially preferably for branched and unbranchedC₁₋₆-alkyl, C₃₋₆-cycloalkyl radicals. Alkenyl stands for branched andunbranched C₂₋₂₀-alkenyl, branched and unbranched C₁₋₂₀-alkyl-O-C₂₋₂₀alkenyl, C₃₋₂₀(—O/S—C₂₋₂₀)₂₋₂₀ alkenyl, aryl-C₂₋₂₀-alkenyl, branched andunbranched heterocyclyl C₂₋₂₀ alkenyl, C₃₋₂₀-cycloalkenyl, preferablyfor branched and unbranched C₂₋₁₂-alkenyl, branched and unbranchedC₁₋₁₂(—O/S—C₂₋₁₂)₂₋₁₂ alkenyl, especially preferably for branched andunbranched C₂₋₆-alkenyl, branched and unbranched C₁₋₆ (—O/S—C₂₋₈)₂₋₈alkenyl radicals; alkynyl stands for branched and unbranchedC₂₋₂₀-alkynyl, branched and unbranched C₃₋₂₀(—O/S—C₂₋₂₀)₂₋₂₀ alkynyl,preferably for branched and unbranched C₂₋₁₂-alkynyl, branched andunbranched C₁₋₁₂ (—O/S—C₂₋₁₂)₂₋₁₂ alkynyl, especially preferably forbranched and unbranched C₂₋₆-alkynyl, branched and unbranchedC₁₋₆(—O/S—C₂₋₈)₂₋₈ alkynyl radicals; cycloalkyl stands for bridged andunbridged C₃₋₄₀-cycloalkyl, preferably for bridged and unbridgedC₃₋₂₆-cycloalkyl, especially preferably for bridged and unbridgedC₃₋₁₅-cycloalkyl radicals; aryl stands for substituted and unsubstitutedmono- or multi-linked phenyl, pentalenyl, azulenyl, anthracenyl,indacenyl, acenaphtyl, fluorenyl, phenalenyl, phenanthrenyl, preferablyfor substituted and unsubstituted mono- or multi-linked phenyl,pentalenyl, azulenyl, anthracenyl, indenyl, indacenyl, acenaphtyl,fluorenyl, especially preferably for substituted and unsubstituted mono-or multi-linked phenyl, pentalenyl, anthracenyl radicals as well astheir partly hydrated derivatives. Heterocyclic compounds can beunsaturated and saturated 3-15-membered mono-, bi- and tricyclic ringswith 1-7 heteroatoms, preferably 3-10-membered mono-, bi- and tricyclicrings with 1-5 heteroatoms and especially preferably 5-, 6- and10-membered mono-, bi- and tricyclic rings with 1-3 heteroatoms.

[0045] In addition, at the alkyl, alkenyl, alkynyl, cycloalkyl, aryl,heteroatoms, heterocyclic compounds, biomolecules or natural substance,0 to 30 (preferably 0 to 10, especially preferably 0 to 5) of thefollowing substituents can occur singly or in combination with oneanother: fluorine, chlorine, bromine, iodine, hydroxyl, amide, ester,acid, amine, acetal, ketal, thiol, ether, phosphate, sulphate,sulphoxide, peroxide, sulphonic acid, thioether, nitrile, urea,carbamate, wherein the following are preferred: fluorine, chlorine,bromine, hydroxyl, amide, ester, acid, amine, ether, phosphate,sulphate, sulphoxide, thioether, nitrile, urea, carbamate and especiallypreferred are: chlorine, hydroxyl, amide, ester, acid, ether, nitrile.

[0046] By directional immobilisation it should herein especially beunderstood that every amino acid sequence is bound to the surface via adefined reactive group or collection of reactive groups. As a result ofthis binding specificity it is achieved that within the limits of theusual entropies the individual amino acid sequences are in anenergetically preferred state so that the amino acid sequencesimmobilised to such an extent are in broadly similar secondary andtertiary structures.

[0047] By the concept “assembly of a plurality of amino acid sequences”it is herein especially understood that each amino acid sequence isimmobilised at a specific location on the surface. Preferably each ofthese locations can be identified. The locations are thus distinctlocations at which respectively one species of amino acid sequence issubstantially immobilised. In other words, there exists a map from whichthe position of each of the immobilised amino acid sequences on thesurfaces can be derived. The individual amino acid sequence canrepresent a plurality of molecules which are however substantiallyidentical in respect of their amino acid sequence, i.e. the type andsequence of the amino acids forming them. The identity of the amino acidsequence is substantially determined by the method of producing theamino acid sequences. It is within the scope of the present inventionthat the amino acid sequences are synthesised in situ on the surface ofthe assembly, wherein all possible forms are feasible here, i.e.,sequential attachment of the individual amino acids forming the aminoacid sequence in the same way as the use of block synthesis techniquesin which groups of amino acids are added together and the individualblocks are then strung together sequentially and the blocks or sequencesthereof are then immobilised or attached to already immobilised aminoacid sequences.

[0048] It is understandable to the person skilled in the art that as aresult of the not always complete yields of the individual synthesissteps or coupling steps, certain heterogeneities can arise in thevarious amino acid sequences in the sense described previously. This canespecially be a problem for syntheses requiring many reaction steps, asis the case with the synthesis of amino acid sequences (per amino acidbuilding block, one coupling reaction and one protective group cleavingand, at the end of the synthesis generally, one reaction for thesimultaneous cleaving of all protective groups of the side chainfunctions). Thus, for example, during the synthesis of one amino acidsequence consisting of 20 amino acid building blocks or 40 amino acidbuilding blocks, and an assumed average yield of 95% for the necessary41 or 81 reaction steps, the predicted theoretical yield is only0.95⁴¹=0.122 (12.2%) or 0.95⁸¹=0.0157 (1.57%). Even for an assumedaverage yield of 99% only 66.2% or 44.3% are obtained for the examplescited above. It thus becomes clear that during the enzymatic reactionsto be studied, as a result of these limitations in addition to thedesired amino acid sequence there are a large number of other amino acidsequences which are distinguished by the absence of one or a pluralityof amino acid building blocks. Precisely these by-products, known to theperson skilled in the art as error or Rumpf sequences, can under certaincircumstances seriously distort the result of the incubation with anenzymatic activity modifying the amino acid sequences arranged on thesurface or make it difficult to interpret the results. For example,during the immobilisation of a substrate for a kinase on or at a surfaceusing the amino groups contained within this substrate, there are aplurality (depending on the number of amino groups present in thecompound to be immobilised) of possibilities for the reaction and thusfor the final orientation of the compound on the surface. If just one ora plurality of these amino groups is required for the effectiveformation of an enzyme/substrate/complex during the subsequentincubation of this immobilised compound (kinase substrate) with abiological fluid containing at least one kinase activity, such anon-specific immobilisation can have the result that only a smallpopulation of the immobilised substrate is anchored in the correctfashion and thus the measurement signal is below the detection limit.Thus, a specific or directional immobilisation is a great advantage. Inthis case, in an immobilisation event the contact between the compoundto be immobilised and the surface on or at which the compound isimmobilised takes place in the same fashion in each case and allcompounds are bound on or to the surface in a defined and predictableorientation.

[0049] The plurality of amino acid sequences consists of at least twodifferent amino acid sequences. It can be provided that the amino acidsequence immobilised at a distinct site reoccurs at another site on thesurface. This can be achieved for example for control purposes.

[0050] The planar surface can be such a surface that is alignedsubstantially two-dimensionally. Especially it is not provided accordingto the present invention that the surface carrying a plurality of aminoacid sequences is a spherical surface or a substantial part of such asurface. During the development of the planar surface it is preferredthat the distinct locations at which respectively one amino acidsequence is localised are not or at least are not substantiallyseparated by a three-dimensional structure from another distinctlocation on the surface.

[0051] All biotolerable, functionalised or functionalisable materialscan be used as materials for the surface or as support materials whichcan carry the assemblies according to the invention within the scope ofthe present invention. These materials can, for example, be present assolid support plates (monolithic blocks), membranes, films or laminates.Suitable materials are polyolefins, such as, for example, polyethylene,polypropylene, halogenated polyolefins (PVDF, PVC etc,) as well aspolytetrafluoroethylene. On the inorganic materials side, for example,ceramic, silicates, silicon and glass can be used. Although non-metallicsupport plates are preferred, it is however also within the scope of thepresent invention to use metallic support materials despite theirtendency to form potentially non-specific adsorption effects. Examplesof such materials are gold or metal oxides, such as titanium oxide forexample.

[0052] Regardless of the material actually selected, wherein glass isparticularly preferred, it is also essential for the present inventionthat the surface is of a non-porous nature and capillary forces do notoccur or do not substantially occur at the surface.

[0053] During the development of the assembly, there are a number ofpossibilities for the design of the surface in the actual sense, i.e.the planar surface carrying the plurality of amino acid sequences. It isfundamentally possible that the surface on which the directionalimmobilisation of the amino acid sequences takes place is at the sametime the support material. However, it is also possible that thereactive surface differs from the support material. Such a scenario isprovided, for example, if the material forming the (planar) surface ispresent in the form of a film, which is then applied to a further basesupport material, not least for stabilisation purposes.

[0054] For purposes of directional immobilisation, especially if thistakes place by covalent bonding of the amino acid sequences on a supportmaterial, the surface of the support plate can be functionalised. Aplurality of successive functionalisations is fundamentally possiblebut, depending on the support material selected, a functionalisation canalso be omitted.

[0055] A first functionalisation which is already suitable to accomplisha covalent bonding of the amino acid sequences to the surface can beaccomplished in the provision of amino and/or carboxyl groups asreactive groups. Such a functionalisation, regardless of the chemicalnature of the reactive groups applied, is also designated herein asfirst functionalisation. Carboxyl groups can be produced by oxidationwith chromic acid, for example, starting from polyolefins as thematerial providing the surface. Alternatively this can also beaccomplished, for example, by high-pressure reaction with oxalylchloride as well as plasma oxidation, radical or light-induced additionof acrylic acid or the like. As a result of base-catalysed eliminationprocesses which lead to double bonds at the surface, halogenatedmaterials such as halogenated polyolefins can result in the productionof both amino- and carboxy-reactive groups, whereby the reactive doublebonds are then carboxy- or amino-functionalised.

[0056] Ceramics, glasses, silicon oxide and titanium oxide can be simplyfunctionalised using substituted silanes available commercially in aplurality such as, for example, aminopropyl triethoxy silane. Supportplates with hydroxyl groups on the surface can be modified by aplurality of reactions. Reactions with biselectrophiles are especiallyadvantageous, such as for example, the direct carboxymethylation withbromacetic acid; acylation with a corresponding amino acid derivativesuch as, for example, dimethylaminopyridine-catalysed carbodiimidecoupling with fluorenyl methoxycarbonyl-3-aminopropionic acid or thegeneration of iso(thio-) cyanates by mono-conversion with correspondingbis-iso(thio)cyanates. An especially advantageous method is the reactionwith carbonyl diimidazole or phosgene or triphosgene or p-nitrophenylchloroformiate or thiocarbonyl diimidazole followed by the reaction withdiamine or simply protected diamines in order to apply amino functionsto the support materials via a stable urethane bond on the surface.

[0057] According to the present invention, it can be provided that theamino acid sequences immobilised on the surface have a spacer. Suchspacers are especially preferred when the amino acid sequences are thesubstrate for enzymatic activities which should occupy a specificspatial structure in order to be thereby accessible for the enzymaticactivity. As a result of using such spacers, herein also designated as“spacer”, the amino acid sequences which should be the actual substratesfor said enzymatic activity or activities, gain additional degrees offreedom and surface phenomena such as adsorption, change in thethermodynamic degrees of freedom etc., will occur. A spacer cansubstantially be any biocompatible molecule that contains at least twofunctional or functionalisable groups. The spacer is inserted in theused state as an element between the surface and the amino acidsequence.

[0058] The following classes of compounds are suitable as spacers:

[0059] Alkanes, branched or unbranched, especially those having a chainlength of C2 to C30, especially C4 to C8;

[0060] Polyethers, i.e., polymers of polyethylene oxide or polypropyleneoxide, wherein the polyethers preferably consist of 1 to 5 polyethyleneoxide units or polypropylene oxide units.

[0061] Polyalcohols, branched or unbranched such as polyglycol andderivatives thereof, such as for exampleO,O′-bis(2-aminopropyl)-polyethylene glycol 500 and 2,2′-(ethylenedioxide)-diethyl amine.

[0062] Polyurethane, polyhydroxy acids, polycarbonates, polyimide,polyamide, polyester, polysulphones, especially those comprising 1-100monomer units, quite especially preferably consisting of 1-10 monomerunits.

[0063] Combinations of the aforesaid alkanes with the aforesaidpolyethers; polyurethanes, polyhydroxy acid, polycarbonates, polyimides,polyamides, polyamino acids, polyesters and polysulphones

[0064] Diamino alkanes, branched or unbranched, preferably those havinga chain length of C2 to C30, quite especially preferably those having achain length of C2 to C8; as examples mention may be made of 1,3-diaminopropane, 1,6-diamino hexane and 1,8-diamino octane, as well as theircombinations with polyethers, preferably with the aforesaid polyethers;such as for example 1,4-bis-(3-aminopropoxy)butane.

[0065] Dicarbonic acids and their derivatives, such as for example,hydroxy-, mercapto, and amino dicarbonic acids, saturated orunsaturated, branched or unbranched, especially C2 to C30 dicarbonicacids, preferably those having a chain length of C2 to C10, quiteespecially preferably those having a chain length of C2 to C6; such asfor example, succinic acid and glutaric acid; and

[0066] Amino acids and peptides, preferably having a length of 1-20amino acid residues, quite especially preferably having a length of 1-3amino acid residues, for example, trimers of lysine, dimers of 3-aminopropionic acid and monomers of 6-amino capronic acid.

[0067] As a result of the fact that the spacer has two functional ends,it is fundamentally possible to select the functionality so that theamino acids to be immobilised on the surface are either immobilised viatheir C-terminus or their N-terminus or via another functional groupingwithin the amino acid sequence to be immobilised. If an immobilisationis to take place via the C-terminus, the functional group of the spaceracting on the C-terminus is preferably an amino group. If the amino acidsequences are to be immobilised by means of the N-terminus to thesurface, the corresponding functional group of the spacer is a carboxylgroup.

[0068] In the assembly according to the invention, it can be providedthat the spacer is a branched spacer. Such branched spacers are alsocalled dendrimer structures or dendrimers for short and are known to theperson skilled in the art. Dendrimer structures for the immobilisationof nucleic acids are described, for example, in Beier, M. & Hoheisel, J.D., 1999, Versatile derivatisation of solid support media for covalentbonding on DNA microchips, 9, 1970-1977. The function of these dendrimerstructures consists in increasing the number of reactive groups per unitarea and thus the signal intensity. Dendrimer structures can be providedwith almost all functional or functionalisable groups which then allowimmobilisation of the amino acid sequences. As a result of using suchdendrimer structures, the number of reactive groups per unit area of theplanar surface can then be increased by a factor of 2 to 100, preferablyby a factor of 2 to 20 and more preferably by a factor of 2 to 10.

[0069] The construction of a dendrimer structure can be accomplished,for example, in the case where the surface is provided with an aminofunctionality by a reaction sequence comprising an acylation withacrylic acid or acrylic acid derivatives such as acrylic acid chlorideor alpha-bromo carbonic acids or alpha-bromo carbonic acid derivativessuch as bromacetyl bromide, Michael addition of suitable polyamines suchas, for example, tetraethylene pentamine, then further acylation withacrylic acid or acrylic acid derivatives such as acrylic acid chlorideor alpha-bromo carbonic acids or alpha-bromo carbonic acid derivativessuch as bromacetyl bromide and further Michael addition of suitablepolyamines. The polyamines are preferably selected such that they arehydrophilic themselves in order to increase the hydrophilic property ofthe surface. An example of such a polyamine is1,4-bis-(3-aminopropoxy)butane.

[0070] In addition to the first functionalisation of the surface, asecond functionalisation can take place, which builds on the firstfunctionalisation. In other words, the reactive group of the surface isextensively functionalised by additional measures. The secondfunctionalisation can take place directly on the functionalised surface,on the surface provided with a spacer or on a dendrimer structure.

[0071] A reason for the second functionalisation can be seen in that, asa result of the amino and carboxyl groups present in the amino acidsequences, thiol functions, imidazole functions and guanido functions,no uniform immobilisation relative to the orientation of the amino acidsequence on the surface can be achieved. A second functionalisationprovides access to further chemoselective reactions in order to achievedirectional immobilisation.

[0072] All those compounds distinguished by a presence ofnon-proteinogenic functional groups are suitable for this secondfunctionalisation. For example, the following compounds may bementioned: maleinimido compounds such as maleinimido amine ormaleinimido carbonic acids; alpha-halo-ketones such as bromo-pyroracemicacid or 4-carboxy-alpha-bromo-acetophenone, alpha-isothiocyanato-ketonessuch as 4-carboxy-alpha-isothiocyanato-acetophenone, aldehydes such ascarboxybenzaldehyde, ketones such as levulinic acid, thiosemicarbazide,thioamides such as succinic acid monothioamide, alpha-bromo-carbonicacids such as bromoacetic acid, hydrazines such as 4-hydrazinobenzoicacid, O-alkylhydroxylamines such as amino-oxy-acetic acid and hydrazidessuch as glutaric acid monohydrazide.

[0073] As a further measure during the development of the assemblyaccording to the invention it can be provided that those sites orregions of the surface not provided with an amino acid sequence areblocked. The blocking ensures that during or after the chemoselectivereaction of the amino acid sequences with the, if necessary,functionalised surfaces, groupings or groups which have not yet reactedbut are still reactive on the surface are inactivated. This blockingreaction is necessary since otherwise the added enzymatic activity orother constituents of the biological sample used react non-specificallywith reactive groups on the surface which are not yet blocked and canthus possibly provide a large background signal. Such non-specificreactions with surfaces are a frequent cause of unfavourablesignal-to-noise ratios in biochemical analyses. Those compounds whichare not sterically demanding, which react very well with the groups tobe blocked and generate surface properties as favourable as possible aresuitable for this blocking. The choice of these compounds will depend onthe type of sample or the interaction partner which interacts with oneof the amino acid sequences. The compound will be configured ashydrophilic if it is known that the enzymatic activity preferably bindsnon-specifically to hydrophobic surfaces and hydrophobic if it is knownthat the enzymatic activity preferably binds non-specifically tohydrophobic surfaces. Thus, it is known to the person skilled in the artthat a biomolecule, such as a protein, for example, requires athree-dimensional, precisely defined structure for the correctbiological function. This tertiary structure is significantly dependenton the environment. Thus, a protein in water, which is a hydrophilicsolvent, has the tendency to conceal all or more accurately, as manygroupings as possible in the interior. If such a protein enters a morehydrophobic environment (hydrophobic surface), folding over or unfoldingof the protein and therefore inactivation can occur. On the other hand,proteins are known which in their natural mode of occurrence are presentinside (hydrophobic) biomembranes Such proteins would fold over oncoming in contact with a hydrophilic surface and thereby denature orbecome inactivated. In such a case a hydrophobic surface is desirable.

[0074] The constituents of the amino acid sequences of the assemblyaccording to the invention are amino acids preferably selected from thegroup comprising the L and D amino acids. The amino acids canfurthermore be selected from the group comprising natural and unnaturalamino acids. A preferred group within each of the previous groups ofamino acids are the corresponding alpha amino acids. The amino acidsequences can consist of a sequence of amino acids from any one of theprevious groups. Thus, for example, a combination of D and L amino acidsis within the scope of the invention in the same way as amino acidsequences which consist either exclusively of D or L amino acids. Theconstituents of the amino acid sequences can furthermore comprisemolecules other than amino acids. Examples herefor are thioxo-aminoacids, hydroxy acids, mercapto acids, dicarbonic acids, diamines,dithioxocarbonic acids, acids and amines. Another form of derivatisedamino acid sequences are the so-called PNAs (peptide nucleic acids).

[0075] The density of the amino acid sequences is 1/cm² to 2000/cm²,wherein the density is preferably 5/cm² to 1000/cm² and quite especiallypreferably 10/cm² to 100/cm². Such densities of distinct locations on asurface which can each contain an amino acid species, can be achievedusing various techniques such as, for example, piezoelectrically drivenpipetting robots, using fine needles made of various materials such aspolypropylene, stainless steel or tungsten or corresponding alloys,using so-called pin-tools which are either slotted needles or areconstructed of a ring containing the substance mixture to be applied anda needle which through the substance mixture contained in this ring,drops this onto the corresponding surface. However, capillariesconnected to a motor-driven spray are also suitable (spotters). Anotherpossibility is to apply the samples to be immobilised using suitablestamps. However, it is also possible to apply the amino acid sequencesto be immobilised by hand by using suitable pipettes or so-calledmultipettes. It is furthermore possible to produce the densities ofdistinct locations given above by direct in situ synthesis of the aminoacid sequences. (M. Stankova, S. Wade, K. S. Lam, M. Lebl; 1994,Synthesis of combinatorial libraries with only one representation ofeach structure, Pept. Res., 7, 292-298, F. Rasoul, F. Ercole, Y. Pham,C. T. Bui, Z. M. Wu, S. N. James, R. W. Trainor, G. Wickham, N.J. Maeji;2000, Grafted supports in solid-phase synthesis, Biopolymers, 55,207-216, H. Wenschuh, R. Volkmer-Engert, M. Schmidt, M. Schulz, J.Schneider-Mergener, U. Reineke; 2000, Coherent membrane supports forparallel microsynthesis and screening of bioactive peptides,Biopolymer., 55, 188-206, R. Frank, 1992, Spot synthesis: an easytechnique for the positionally addressable, parallel chemical synthesison a membrane support, Tetrahedron, 48, 9217-9232; A. Kramer and J.Schneider-Mergener, Methods in Molecular Biology, vol. 87: CombinatorialPeptide Library Protocols, p. 25-39, edited by: S. Cabilly; Humana PressInc., Totowa, N.J.; Töpert, F., Oires, C., Landgraf, C., Oschkinat, H.and Schneider-Mergener, J., 2001, Synthesis of an array comprising 837variants of the hYAP WW protein domain, Angew. Chem. Int. Ed., 40,897-900, S. P. A. Fodor, J. L. Read, M. C. Pirrung, L. Stryer, A. T. Lu,D. Solas; 1991, Light-directed spatially addressable parallel chemicalsynthesis, Science, 251, J. P. Pellois, W. Wang, X. L. Gao; 2000,Peptide synthesis based on t-Boc chemistry and solution photogeneratedacids, J. Comb. Chem. 2, 355-360).

[0076] In a preferred embodiment of the assembly according to theinvention and its various uses and applications, it is provided that thevarious amino acid sequences are substrates or possible substrates ofenzymatic activities, which are contained in the samples as interactionpartners towards which the assembly according to the invention isexposed. Enzymatic activities should generally be understood herein asthose enzymatic activities which are characterised in that they transferan atom group, a molecule or a molecular group to a molecule. Enzymaticactivities should herein especially be understood as kinases,sulphotransferases, glycosyl transferases, acetyl transferases, farnesyltransferases, palmityl transferases, phosphatases, sulphatases,esterases, lipases, acetylases and proteases. The enzymatic activitywill accordingly change if necessary one or a plurality of amino acidsequences of the assembly, that is one or a plurality of amino acids onthe chip, with respect to its molecular weight. Such a change in themolecular weight can comprise a decrease or an increase in the same, andmay involve further changes to the physicochemical properties of theamino acid sequences or the distinct locations at which respectively onespecies of amino acid sequence is located.

[0077] Various techniques known to the person skilled in the art can beused to detect whether a binding event takes place at one or a pluralityof the various amino acid sequence species and, insofar as theinteraction partner of the amino acid sequence is an enzymatic activityor carries this, whether an enzymatic conversion takes place at therespective amino acid sequence. Thus it is possible to trace a cleavingreaction. e.g. mediated by a protease, by a change in the fluorescenceof a suitable substrate molecule bound to the surface. In principle, allreactions during which the molecule bound to the surface is changed inmolecular weight by transfer of other molecules (co-substrates) can betraced by incorporating a radioactive label into the co-substrate. Forthis purpose the radioactivity incorporated into the modified moleculebound to the surface must be quantified after the reaction. With the aidof such radioactive labels, all transferases such as, for example,kinases, acetyl transferases, farnesyl transferases and glycosyltransferases can be characterised with reference to enzymatic activity.Alternatively, reactive groups which have been produced from therespective enzymatic reaction at the respective amino acid sequence andwhich were not previously present can be detected by means of subsequentspecific reactions. For example, a mercapto function obtained after anenzymatic reaction can be detected by means of a following reaction withEllman reagent.

[0078] It is within the scope of the present invention that the assemblycomprises a certain number of different amino acid species. In this caseit can be provided that the same amino acid sequence is present at aplurality of distinct locations on the surface or the support material.Thus, on the one hand, an internal standard can be achieved and on theother hand, however, possible edge effects can be represented andrecorded.

[0079] A further development of the invention provides that at least twoor a plurality of assemblies are joined together such that between thetwo assemblies there is only a very small gap into which the amino acidsequences of the two assemblies extend (see FIG. 12B). This developmentis herein also called a support assembly. This opens up a possibilityfor carrying out a plurality of tests using a very small sample volume.The width of the gap is 2 mm, preferably 0.5 mm and preferably less than0.1 mm. This gives a liquid volume of less than 100 nL relative to asurface area of 1 mm². It is within the scope of the present inventionthat the assemblies forming the support assembly differ in respect ofdevelopment. These differences can consist in the fact that the aminoacid sequences are all or partly different. It is furthermore possiblethat the amino acid sequences in the different assemblies are arrangedcompletely or partly at other distinct locations.

[0080] The assembly according to the invention offers a number ofpossible applications. One such application is the determination of thesubstrate specificity of the enzymatic activity (FIG. 12, compound C).In this case, the procedure is that in a first step an assemblyaccording to the invention or chip is prepared and this is brought intocontact, and if necessary incubated, with a sample containing therespective enzymatic activity. The reaction is then detected between oneor a plurality of amino acid sequences present on the assembly (FIG.12A, compounds B₁-B₃ or FIG. 12B, compounds B₁-B₅) and the enzymaticactivity (FIG. 12, compound C), wherein the detection methods describedabove can be used. As a result of the arrangement of the different aminoacid sequence species at distinct locations, a reaction event of aspecific amino acid sequence or amino acid sequence species (both termsare used herein synonymously) can thus be uniquely assigned to aspecific location and the substrate specificity of the enzymaticactivity can be determined therefrom (see FIGS. 8, 9 and 10).

[0081] Starting from the substrate specificity, for example, theinfluence of various substances on the respective reaction can beinvestigated. For example, depending on the respective substrate, anenzymatic activity can undergo a specific modification by a factor addedto the reaction formulation containing the enzymatic activity, forexample, a low-molecular compound.

[0082] Another application of the assembly according to the invention isin displaying the differential analysis of the enzymatic activities of asample. An especially important sample in this respect is the proteomeof a cell with reference to which this aspect of the invention isexplained in the following. In this case, unlike the applicationdescribed previously, attention is not focussed on the specificity of anindividual enzymatic activity with respect to the amino acid speciespresent on the surface but rather to a certain extent on aninstantaneous snapshot of the enzymatic activities in a sample withrespect to the various amino acid sequence species of the assembly. Thisinstantaneous snapshot was made under certain conditions which prevailedat the time the sample was taken. In the case of the proteome, this canfor example be the state after exposure of the cell from which thesample was obtained, to a certain compound. One or a plurality offurther samples are then taken wherein the conditions which prevailed atthe time of sampling are changed, for example, the cell was no longerexposed to said compound and an analysis is then made. Depending on theselected method of detection, the result of the reaction event is thencompared under the different conditions and from this it can bedetermined whether and, if so, to what extent the pattern from therespective reaction event has changed. On the other hand, such anassembly of amino acid sequences can also be used to compare biologicalsamples one with the other such as cell lysates, for example, orbiological fluids of one species or different species by means ofpattern recognition or to catalogue these biological samples by means ofthe pattern obtained. Such a pattern is then used in the transferredsense as a fingerprint of the biological sample studied. Thus, themethod according to the invention can be used for identification orindividualisation. The identification can take place on differentsystematic levels, i.e., the allocation of suitably studied sample to astrain, a class, an order, a family, a genus, or a type. Furthermore,the identification can also take on the level of the type betweenindividuals of the same type or race. For example, this method can beused in forensic science. A further application of the method can beseen in determining, diagnosing or predicting pathological states suchas cancer or a pattern of enzymatic activity changed compared with thenorm (both quantitatively and qualitatively).

[0083] The present invention is now explained with reference to thefollowing drawings and examples from which further features, exemplaryembodiments and advantages can be obtained. In the figures:

[0084]FIG. 1. shows the result of incubating a differently modifiedglass surface with different kinases;

[0085]FIG. 2 shows the result of incubating various modified surfaceswith protein kinase A;

[0086]FIG. 3 shows the result of time-dependent incubation of variousmodified surfaces with protein kinase A;

[0087]FIG. 4 shows the result of incubating a differently modified glasssurface with different concentrations of protein kinase A FIG. 5 showsthe result of incubating a modified glass surface with protein kinase A;

[0088]FIG. 6: shows the result of incubating a modified glass surface(11760 spots) with a kinase;

[0089]FIG. 7: shows the result of incubating a modified glass surface(960 spots) with a kinase;

[0090]FIG. 8: shows the result of incubating a glass surface modifiedwith a set of potential substrate peptides and corresponding controlpeptides with protein kinase C;

[0091]FIG. 9: shows the result of incubating a glass surface modifiedwith a set of potential substrate peptides and corresponding controlpeptides with protein kinase A;

[0092]FIG. 10: shows the result of incubating a glass surface modifiedwith a set of potential substrate peptides with protein kinase A;

[0093]FIG. 11: shows an overview of various chemoselective reactions;and

[0094]FIG. 12: shows a schematic structure of various embodiments of anassembly of compounds directionally immobilised on a support surface.

[0095] The figures are now described in detail.

[0096]FIG. 1. The kinase substrates given in parentheses (modified atthe N-terminus with the dipeptide cysteinyl-β-alanine) were coupled to amaleinimido-functionalised glass surface by a Michael addition (Example1). Peptides for which the serine amino acid to be phosphorylated wasexchanged for the non-phosphorylatable amino acid alanine were used asnegative controls. The glass surface was first pre-incubated for 10minutes using 10 mL of 100 μM ATP solution in 50 mM of sodium phosphatebuffer pH 7.5. The corresponding kinases were then spotted on togetherwith ATP/γ³²P-ATP mixture (1 μL, 5 U/mL in each case) and incubated for30 minutes at 25° C. (Example 29). The phosphorylation of thecorresponding peptides was detected using a FUJIFILM PhosphorImager.

[0097]FIG. 2. The peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly-NH₂ and thecontrol peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly-NH₂, each modified at theN-terminus with the dipeptide cysteinyl-β-alanine, were coupled to amaleinimido-functionalised surface by a Michael addition (Example 1;maleinimidobutyryl-β-alanine-functionalised cellulose as well asmaleinimidobutyryl-β-alanine-functionalised, modified polypropylenemembranes). The surfaces thus modified were first pre-incubated for 10minutes using 10 mL of 100 μM ATP solution in 50 mM of sodium phosphatebuffer pH 7.5. Protein kinase A was then spotted on together withATP/γ³²P-ATP mixture (100 μL/mL; 100 μCi/mL) (1 μL. 2 U/mL in each case)and incubated for 30 minutes at 25° C. (Example 30). The phosphorylationof the corresponding peptides was detected using a FUJIFILMPhosphorImager. The signal intensity of the respective spot is givenbelow the figures. It is clear that under the selected experimentalconditions in the case of modified cellulose or polypropylene surfaces,in principle only non-specific binding of ATP or kinase to the peptidesis measured. In the case of the modified glass surfaces, however, thesignal for the substrate amino acid sequence is a factor of 4-5 largerthan the signal for the corresponding control amino acid sequence.

[0098]FIG. 3. The peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly-NH₂, modified atthe N-terminus with the dipeptide cysteinyl-β-alanine, was coupled tomaleinimido-functionalised surfaces by a Michael addition(maleinimido-functionalised glass surface, Sigma, Silane-Prep™, S4651;as well as maleinimidobutyryl-β-alanine-functionalised, modifiedpolypropylene membranes). The modified glass surfaces were firstpre-incubated for 10 minutes using 20 mL of 100 μM ATP solution in 50 mMof sodium phosphate buffer pH 7.5. Protein kinase A was then spotted ontogether with ATP/γ³²P-ATP mixture (100 μL/mL; 100 μCi/mL) (1 μL, 2 U/mLin each case) and incubated for the given time at 25° C. (Example 31).The phosphorylation of the corresponding peptides was detected using aFUJIFILM PhosphorImager. The signal intensity of the respective spot isgiven below the figures. It is clear that under the selectedexperimental conditions in the case of modified polypropylene surfaces,in principle only non-specific binding of ATP or kinase to the peptidesis measured. In the case of the modified glass surfaces, however, aclear time dependence can be identified for the kinase-mediatedincorporation of radioactivity into the substrate amino acid sequence.

[0099]FIG. 4. The control peptide Leu-Arg-Arg-Ala-Ala-Leu-Gly-NH₂, thepeptide Leu-Arg-Arg-Ala-Ala-Leu-Gly-NH₂ and the synthesis raw productLeu-Arg-Arg-Ala-Ala-Leu-Gly-NH₂, each modified at the N-terminus withthe dipeptide cysteinyl-β-alanine, were coupled to amaleinimido-functionalised glass surface by a Michael addition (Example1). The modified glass surface was first pre-incubated for 10 minutesusing 10 mL of 100 μM ATP solution in 50 mM of sodium phosphate bufferpH 7.5. The modified glass surface was then covered with a cover glassand Protein kinase A (1 U/mL or 10 U/mL) together with ATP/γ³²P-ATPmixture (100 μL/mL; 100 μCi/mL) was then inserted into the intermediatespace formed thereby by means of capillary force (Example 25). Afterincubation for 30 min at 25° C. the phosphorylation of the correspondingpeptides was detected using a FUJIFILM PhosphorImager. The signalintensity of the respective spot is given below the figures. It is clearthat under the selected experimental conditions the signal intensity forthe purified amino acid sequence is 500% higher than that for thesynthesis product. The signal intensity for the purified amino acidsequence is approximately 300 times higher than that for thecorresponding control amino acid sequence. Together with theapproximately ten times lower quantity of activity required for acomparable signal (compared with cellulose surfaces), an improvement insignal by a factor of 3000 is thus obtained.

[0100]FIG. 5. The control peptide Leu-Arg-Arg-Ala-Ala-Leu-Gly-NH₂, andthe peptide Leu-Arg-Arg-Ala-Ala-Leu-Gly-NH₂, each modified at theN-terminus with the dipeptide cysteinyl-β-alanine, were coupled to amaleinimido-functionalised glass surface by a Michael addition (Example1). The modified glass surface was first pre-incubated for 10 minutesusing 10 mL of 100 μM ATP solution in 50 mM of sodium phosphate bufferpH 7.5. The modified glass surface was then covered with a cover glassand Protein kinase A (10 U/mL) together with ATP/γ³²P-ATP mixture (100μM/mL; 100 μCi/mL) was then inserted into the intermediate space formedthereby by means of capillary force. After incubation for 30 min at 25°C. the phosphorylation of the corresponding peptides was detected usinga FUJIFILM PhosphorImager. The signal intensity of the respective spotis given below the figures. It is clear that under the selectedexperimental conditions the signal intensity for the substrate aminoacid sequence is 800% higher than that for the control product.

[0101]FIG. 6. A glass surface modified with the peptideLeu-Arg-Arg-Ala-Ser-Leu-Gly-thioamide (see Example 24) amino acidsequence was dissolved in 200 mM sodium phosphate buffer pH 5.5 and atroom temperature respectively 1 nL of this solution in an assembly of 70rows and 168 gaps (total 11760) was applied to thebromoketone-functionalised glass surfaces (Example 10) using aNanoPlotter from Gesim. The spot-to-spot distance was 0.3 mm. The glasssurfaces thus treated were then subjected to microwave treatment for 2min and then incubated for 3 hours at room temperature. Pre-incubationwas then carried out for 10 minutes using 10 ml of 100 μM ATP solutionin 50 mM of sodium phosphate buffer pH 7.5. The modified glass surfacewas then covered with a second glass surface and Protein kinase A (10U/mL) together with ATP/γ³²P-ATP mixture (100 μM/mL; 100 μCi/mL) wasthen inserted into the intermediate space formed thereby by means ofcapillary force (see-Example 31). After incubation for 30 min at 25° C.the phosphorylation of the corresponding peptides was detected using aFUJIFILM PhosphorImager. It is clear that on the one hand, the linkingof the immobilised kinase substrate is tolerated by the protein kinase Aand on the other hand, the modification of the glass surfaces takesplace uniformly and without larger fluctuations in the immobilisationdensity. It is furthermore clear that the resolution of thePhosphorImager used here is sufficient to analyse more than 11000measurement points per biochip.

[0102]FIG. 7. A glass surface modified with the peptideDpr(Aoa)-Leu-Arg-Arg-Ala-Ser-Leu-Gly-NH₂ (see Example 22) was firstpre-incubated for 10 minutes using 10 ml of 100 μM ATP solution in 50 mMof sodium phosphate buffer pH 7.5. The modified glass surface was thencovered with a second glass surface and Protein kinase A (10 U/mL)together with ATP/γ³²P-ATP mixture (100 μM/mL; 100 μCi/mL) was theninserted into the intermediate space formed thereby by means ofcapillary force. After incubation for 30 min at 25° C. thephosphorylation of the corresponding peptides was detected using aFUJIFILM PhosphorImager (see Example 14). It is clear that on the onehand, the linking of the immobilised kinase substrate is tolerated bythe protein kinase A and on the other hand, the modification of theglass surfaces takes place uniformly and without larger fluctuations inthe immobilisation density. It is furthermore clear that the resolutionof the PhosphorImager used here is sufficient to analyse more than 950measurement points per biochip.

[0103]FIG. 8. Precisely 43 serine and/or threonine-containing peptides(potential substrate peptides for kinases) and the corresponding controlpeptides, each modified at the N-terminus with the dipeptidecysteinyl-β-alanine, were coupled to a maleinimido-functionalised glasssurface by a Michael addition (Example 18). In the control peptides theserine and/or threonine residues were replaced by alanine, the sequenceremaining otherwise the same. The application was carried out using aNanoPlotter from Gesim. The spot-to-spot distance was 1 mm and 0.8 nL ofa peptide solution in 100 mM PBS buffer pH 7.8, containing 20% glycerin,was applied per spot. The peptide assembly is shown in FIG. 8A. Here afilled circle represents a serine- or threonine-containing potentialsubstrate peptide and an open circle stands for a control peptide. Threeidentical subarrays were applied to the glass surface. The numbering ofthe spots can be seen in FIG. 8C, the sequences of the peptides used areobtained from Example 18. After application of the peptides, themodified glass surface was first pre-incubated for 10 minutes using 10ml of 100 μM ATP solution in 50 mM of sodium phosphate buffer pH 7.5.The modified glass surface was then covered with a second glass surfaceand Protein kinase C (10 U/mL) together with ATP/γ³²P-ATP mixture (100μM/mL; 100 μCi/mL) was then inserted into the intermediate space formedthereby by means of capillary force. After incubation for 30 min at 25°C. the phosphorylation of the corresponding peptides was detected usinga FUJIFILM PhosphorImager (Example 38). The resulting picture is shownin FIG. 8B. The spots having higher signal intensity in all threesubarrays were assigned to the corresponding peptides phosphorylated bythe protein kinase C. Their primary structures are shown in FIG. 8D. Itis clear that peptides known as protein kinase C substrates (substratepeptide No. 3, 23, 27, 41, 43) and other peptides not described assubstrates for protein kinase C are recognised and phosphorylated bythis kinase on the modified glass surface. It is thus clear that such anassembly is suitable for characterising the substrate specificity of akinase, such as protein kinase C, for example.

[0104]FIG. 9. Precisely 43 serine and/or threonine-containing peptides(potential substrate peptides for kinases) and the corresponding controlpeptides, each modified at the N-terminus with the dipeptidecysteinyl-β-alanine, were coupled to a maleinimido-functionalised glasssurface by a Michael addition (Example 18). In the control peptides theserine and/or threonine residues were replaced by alanine, the sequenceremaining otherwise the same. The application was carried out using aNanoPlotter from Gesim. The spot-to-spot distance was 1 mm and 0.8 nL ofa peptide solution in 100 mM PBS buffer pH 7.8, containing 20% glycerin,was applied per spot. The peptide assembly is shown in FIG. 9A. Here afilled circle represents a serine- or threonine-containing potentialsubstrate peptide and an open circle stands for a control peptide. Threeidentical subarrays were applied to the glass surface. The numbering ofthe spots can be seen in FIG. 9C, the sequences of the peptides used areobtained from Example 18. After application of the peptides, themodified glass surface was first pre-incubated for 10 minutes using 10ml of 100 μM ATP solution in 50 mM of sodium phosphate buffer pH 7.5.The modified glass surface was then covered with a second glass surfaceand Protein kinase A (10 U/mL) together with ATP/γ³²P-ATP mixture (100μM/mL; 100 μCi/mL) was then inserted into the intermediate space formedthereby by means of capillary force. After incubation for 30 min at 25°C. the phosphorylation of the corresponding peptides was detected usinga FUJIFILM PhosphorImager (Example 39). The resulting picture is shownin FIG. 9B. The spots having higher signal intensity in all threesubarrays were assigned to the corresponding peptides phosphorylated bythe protein kinase A. Their primary structures are shown in FIG. 9D. Itis clear that with one exception, all peptides on the modified glasssurface are recognised and phosphorylated by protein kinase A whichcarry two arginine residues in position −2 and −3 (N-terminal) toserine. The sequence motif RRxS is described as a preferred substratemotif for protein kinase A (A. Kreegipuu, N. Blom, S. Brunak, J. Jarv,1998, Statistical analysis of protein kinase specificity determinants,FEBS Lett., 430, 45-50). The peptide 83 is probably not phosphorylatedbecause of the excessive N-terminal localisation of the substrate motif.It is thus clear that such an assembly is suitable for characterisingthe substrate specificity of a kinase, such as protein kinase A, forexample.

[0105]FIG. 10. Precisely 79 peptides, each modified at the N-terminuswith the dipeptide amino-oxyacetic acid-β-alanine, were coupled to analdehyde-functionalised glass surface by a Michael addition (Example20). The application was carried out using a NanoPlotter from Gesim. Thespot-to-spot distance was 1.5 mm and 0.8 nL of a peptide solution inDMSO was applied per spot. The 13-mer peptides overlap with respectively11 amino acid residues and together completely cover the primarystructure of MBP, that is together they form a scan through the myelinbasic protein (MBP) from bos taurus (SWISSPROT Accession number PO₂₆₈₇).The primary structure of MBP is shown in FIG. 10C. For the residuesshown in bold print a phosphorylation by protein kinase A was describedin the prior art (A. Kishimoto, K. Nishiyama, H. Nakanishi, Y. Uratsuji,H. Nomura, Y. Takeyama, Y. Nishizuka, 1985, Studies on thephosphorylation of myelin basic protein by protein kinase C andadenosine 3′:5′-monophosphate-dependent protein kinase, J. Biol. Chem.,260, 12492-12499). The 13-mer peptides in the scan show a sequence shiftof two amino acids. The peptide assembly is shown in FIG. 10B. Thus,peptide No. 1 represents the amino acid sequence 1-13 of the primarystructure of MBP, peptide No. 2 represents the amino acid sequence 3-15of the primary structure of MBP, etc. Three identical subarrays wereapplied to the glass surface. One of these subarrays is shown in FIG.10A. After application of the peptides, the modified glass surface wasfirst pre-incubated for 10 minutes using 10 ml of 100 μM ATP solution in50 mM of sodium phosphate buffer pH 7.5. The modified glass surface wasthen covered with a cover glass and Protein kinase A (10 U/mL) togetherwith ATP/γ³²P-ATP mixture (100 μM/mL; 100 μCi/mL) was then inserted intothe intermediate space formed thereby by means of capillary force (seeExample 40). After incubation for 30 min at 25° C. the phosphorylationof the corresponding peptides was detected using a FUJIFILMPhosphorImager. The resulting picture is shown in FIG. 10A. The spotshaving higher signal intensity in all three subarrays were assigned tothe corresponding peptides phosphorylated by the protein kinase A. Theirprimary structures are shown in FIG. 10D. It is clear that most of thepeptides on the modified glass surface are recognised and phosphorylatedby protein kinase A which was also found in the experiment carried outin solution (A. Kishimoto, K. Nishiyama, H. Nakanishi, Y. Uratsuji, H.Nomura, Y. Takeyama, Y. Nishizuka, 1985, Studies on the phosphorylationof myelin basic protein by protein kinase C and adenosine3′:5′-monophosphate-dependent protein kinase, J. Biol. Chem., 260,12492-12499).

[0106]FIG. 11. Shows an overview of various chemoselective reactionsaccording to the prior art: A) aldehyde (R⁴=H) or ketone (R⁴ not H) andamino-oxy compounds react to give oximes, B) aldehyde (R⁴=H) or ketone(R⁴ not H) and thiosemicarbazides react to give thiosemicarbazones, C)aldehyde (R⁴=H) or ketone (R⁴ not H) and hydrazides react to givehydrazones, D) aldehyde (R⁴=H) or ketone (R⁴ not H) and 1,2-aminothiolsreact to give thiazolines (X=S) or 1,2-amino alcohols to give oxazolines(X=O) or 1,2-diamines react to give imadazolines (X=NH), E)thiocarboxylates and α-halocarbonyls react to give thioesters, F)thioesters and β-aminothiols react to give β-mercaptoamides, F)mercaptane and maleinimide react to give succinimides. The radical R¹ inthis case represents alkyl, alkenyl, alkynyl, cycloalkyl or arylradicals or heterocyclic compounds or surfaces and the radicals R⁴-R⁶represent alkyl, alkenyl, alkynyl, cycloalkyl or aryl radicals orheterocyclic compounds or surfaces or H, D or T, wherein alkyl standsfor branched and unbranched C₁₋₂₀-alkyl, C₃₋₂₀-cycloalkyl, preferablyfor branched and unbranched C₁₋₁₂-alkyl, C₃₋₁₂-cycloalkyl, andespecially preferably for branched and unbranched C₁₋₆-alkyl,C₃₋₆-cycloalkyl residues. Alkenyl stands for branched and unbranchedC₂₋₂₀ alkenyl, branched and unbranched C₁₋₂₀-alkyl-C₂₋₂₀-alkenyl,C₂₋₂₀-(—O/S—C₂₋₂₀)₂₋₂₀-alkenyl, branched and unbranchedheterocyclyl-C₂₋₂₀-alkenyl, C₃₋₂₀-cycloalkenyl, preferably for branchedand unbranched C₂₋₁₂-alkenyl, branched and unbranchedC₁₋₃₂-(—O/S—C₂₋₁₂)₂₋₁₂-alkenyl, especially preferably for branched andunbranched C₂₋₁₂-alkenyl, branched and unbranchedC₁₋₆-(—O/S—C₂₋₈)₂₋₈-alkenyl residues; alkynyl stands for branched andunbranched C₂₋₂₀ alkynyl, branched and unbranchedC₁₋₂₀-(—O/S—C₂₋₂₀)₂₋₂₀-alkynyl, preferably for branched and unbranchedC₂₋₁₂-alkynyl, branched and unbranched C₁₋₁₂-(—O/S—C₂₋₁₂)₂₋₁₂-alkynyl,especially preferably for branched and unbranched C₂₋₆-alkynyl, branchedand unbranched C₁₆-(—O/S—C₂₋₈)₂₋₈-alkynyl radicals; cycloalkyl standsfor bridged and unbridged C₃₋₄₀ cycloalkyl, preferably for bridged andunbridged C₃₋₂₆ cycloalkyl, especially preferably for bridged andunbridged C₃₋₁₅ cycloalkyl radicals; aryl stands for substituted andunsubstituted, mono- or multi-linked phenyl, pentalenyl, azulenyl,anthracenyl, indacenyl, acenaphtyl, fluorenyl, phenalenyl,phenanthrenyl, preferably substituted and unsubstituted, mono- ormulti-linked phenyl, pentalenyl, azulenyl, anthracenyl, indenyl,indacenyl, acenaphtyl, fluorenyl, especially preferably for substitutedand unsubstituted, mono- or multi-linked phenyl, pentanenyl, anthracenylresidues, and their partly hydrated derivatives. Heterocyclic compoundscan be saturated and unsaturated 3-15-membered mono-, bi- and tricyclicrings with 1-7 heteroatoms, preferably 3-10-membered mono-, bi- andtricyclic rings with 1-5 heteroatoms and especially preferably 5-, 6-and 10-membered mono-, bi- and tricyclic rings with 1-3 heteroatoms.

[0107] In addition, at the alkyl, alkenyl, alkynyl, cycloalkyl, aryl,heteroatoms, heterocyclic compounds, biomolecules or natural substance 0to 30 (preferably 0 to 10, especially preferably 0 to 5) of thefollowing substituents can occur singly or in combination with oneanother: fluorine, chlorine, bromine, iodine, hydroxyl, amide, ester,acid, amine, acetal, ketal, thiol, ether, phosphate, sulphate,sulphoxide, peroxide, sulphonic acid, thioether, nitrile, urea,carbamate, wherein the following are preferred: fluorine, chlorine,bromine, hydroxyl, amide, ester, acid, amine, ether, phosphate,sulphate, sulphoxide, thioether, nitrile, urea, carbamate and especiallypreferred are: chlorine, hydroxyl, amide, ester, acid, ether, nitrile.

[0108]FIG. 12. Shows a schematic diagram of various embodiments of theincubation of an assembly of compounds (B₁-B₅) on the surface of asupport with an agent (enzymatic activity C) which is capable ofreducing or increasing the molecular weight under the given conditionsfor one or a plurality of compounds in the immobilised state. The linkbetween the surface and the immobilised compounds (A) should be covalentand regioselective. Fig. A shows an embodiment in which the agent C isapplied to the surface. Fig. B however shows an embodiment in which theagent C is applied between two surfaces facing one another which caneither contain the same or different assemblies of immobilisedcompounds.

[0109] The following examples relate to the functionalisation of glasswhose surface is required as a surface for an immobilisation (Examples 1to 14), the immobilisation of various peptides provided with a reactivegroup on a surface (Examples 15 to 24) and the analysis ofkinase-mediated peptide modification using the immobilised peptidesaccording to the invention (Examples 25-34). The abbreviations listedbelow are used:

[0110] Ala, A L-alanine

[0111] Aoa, O Amino oxyacetic acid

[0112] Arg, R L-arginine

[0113] Asn, N L-asparagine

[0114] Asp, D L-asparaginic acid

[0115] ATP Adenosine-5′-triphosphate

[0116] βAla,B,BAL β-alanine, 3-aminopropionic acid

[0117] Boc Tertiary butoxycarbonyl

[0118] Cit L-citrulline

[0119] Cys, C L-cysteine

[0120] DCM Dichloromethane

[0121] DIC N,N′-diisopropyl carbodiimide

[0122] DIPEA N,N′-diisopropyl ethylamine

[0123] DMF N,N′-dimethyl formamide

[0124] DMF N,N′-dimethyl formamide

[0125] DMSO Dimethyl sulphoxide

[0126] EGTA Ethylene glycol-bis-(2-aminoethyl)-N,N,N1,N′-tetracetic acid

[0127] Et Ethyl

[0128] Fmoc 9-fluorenyl methoxycarbonyl

[0129] Gln, Q Glutamine

[0130] Glu, E L-glutaminic acid

[0131] Gly, G Glycine

[0132] HBTU O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyl uroniumhexafluorophosphate

[0133] His, H L-histidine

[0134] HPLC High-performance liquid chromatography

[0135] Ile, 1 L-isoleucine

[0136] L Litre

[0137] Leu, L L-leucine

[0138] Lys, K L-lysine

[0139] M Molar

[0140] MBHA Methylbenzhydryl amine

[0141] MBP Myelin basic protein

[0142] MeOH Methanol

[0143] Met, M L-methionine

[0144] mL Millilitre

[0145] mM Millimolar

[0146] mRNA Messenger RNA

[0147] nL Nanolitre

[0148] Phe, F L-phenylamine

[0149] Pbf 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulphonyl

[0150] Pro, P L-proline

[0151] PTFE Polytetrafluoroethylene

[0152] PVC Polyvinyl chloride

[0153] PVDF Polyvinyl difluoride

[0154] RNA Ribonucleic acid

[0155] RP Reversed-phase

[0156] RT Room temperature

[0157] SDS Sodium lauryl sulphate

[0158] Ser, S L-serine

[0159] tBu Tertiary butyl

[0160] TFA Trifluoroacetic acid

[0161] THF Tetrahydrofuran

[0162] Thr, T L-threonine

[0163] Tris 2-amino-2-hydroxymethyl-1,3-propanediol

[0164] Trp, W L-tryptophan

[0165] Tween20 Polyoxyethylene-sorbitant-monolaurate (trademark of AtlasChemie)

[0166] Tyr, Y L-tyrosine

[0167] U Unit

[0168] Val, V L-valine

[0169] The following reagents and solvents were used:

[0170] Bromine, tert-butyl methyl ether, 1,3-diisopropyl carbodiimide,N,N-diisopropyl ethylamine glacial acetic acid, glycerin, urea, 40%hydroxyamine solution, piperidine, triethylamine, dichloromethane,diethylether, N,N-dimethylformamide, ethanol, methanol andtetrahydrofuran come from Merck Eurolab (Darmstadt, Germany). Oxalylchloride, sodium thiocyanate, trifluroacetic acid, dimethyl sulphoxide,thioacetamide, Lawessons reagent, formic acid and thiourea were obtainedfrom Fluka (Deisenhofen, Germany). Adenosine-5′-triphosphate,2-amino-2-hydroxymethyl-1,3-propanediol hydrochloride, sodium chloride,magnesium chloride, 1,4-dithio-DL-threitol, sodium lauryl sulphate,polyoxyethylene soribitant monolaurate and ethylene glycolbis-(2-aminoethyl)-N,N,N′,N′-tetracetic acid come from sigma(Taufkirchen, German). The Rink amide MBHA resin,(benzatriazol-1-yl)-N,N,N′,N′-tetramethyl uronium hexafluorophosphate aswell as all Fmoc amino acid pentafluorophenyl esters were obtained fromNovabiochem (Bad Soden, Germany).

[0171] Whatman 50 cellulose membranes (Whatman Maidestone, UK) were usedfor the SPOT synthesis.

[0172] Chromatography and Physical Data:

[0173] RP-18-HPLC-MS analyses were carried out by chromatography using aHewlett Packard Series 1100 system (G1322A degasser, G1311A quaternarypump, G1313A automatic sampler, G1316A thermostatically controlledcolumn box, G1314 variable UV detector) and coupled ESI-MS (Finnigan LCQIon Trap Mass Spectrometer). The separation was carried out using RP-18column material (Vydac 218 TP5215, 2.1×150 mm, 5 μm, C18, 300 A withprecolumn) at 30° C. and a flux of 0.3 mL/min using a linear gradientfor all chromatograms (5-95% B within 25 min, wherein A: 0.05% TFA inwater and B: 0.05% TFA in CH₃CN). The UV detection was carried out atλ=220 mm).

[0174] Preparative HPLC was carried out using a Merck/Hitachi system(L-6250 quaternary pump, L-/400 variable UV detector, D-7000 interface,Software: HPLC Systemanager D-7000 for NT 4.0) using a Merck Eurolabcolumn (LiChrospher 100, RP18, 10×250 mm) at a solvent flux of 6.0mL/min. The solvent system used comprised components A (H₂O/0.1 vol. %TFA) and B (CH₃CN/0.1 vol. % TFA).

[0175] Equipment for Producing the Soluble Peptides:

[0176] The peptides used for immobilisation were synthesised fromC-terminal peptide amides using a “Syro” parallel automatic synthesissystem (MultiSynTech, Witten, Germany) using the standard Fmoc protocolon Rink amide MBHA resin. After cleaving from the resin and separatingall the protective groups all the peptides obtained were analysed usingHPLC-MS and showed the desired molecular ion signals. After subsequentHPLC purification the peptides were lyophilised and stored at −20° C.

[0177] The peptides used for the immobilisation (13-mer peptides of theproteins MBP, casein, Histon H1) were produced automatically using thestandard SPOT synthesis method using an Autospot AMS 222 (Abimed,Langenfeld, Germany) using Autospot XL Ver.2.02 control software. Thewashing steps were carried out in stainless steel dishes (Merck Eurolab)which were moved on a tilting table.

EXAMPLES Example 1 Maleinimido Functionalisation of AminopropylsilylatedGlass Surfaces

[0178] 4-maleinimidobutyric acid (Fluka, 63183) was dissolved to 0.3M inDMF. The resultant mixture was activated by adding a half equivalent ofdi-iso-propyl carbodiimide (DIC) for 15 min at room temperature.Aminopropylsilylated glass surfaces purified with compressed air(2.5×7.5 cm; Sigma, Silane-Prep™, S4651) were coated with themaleinimidobutyric acid anhydride solution thus obtained and incubatedfor three hours at room temperature. The glass surfaces thus treatedwere then washed five times using respectively 30 mL of DMF for 3minutes each at room temperature. After washing three times for threeminutes each using respectively 30 mL of dichloromethane (DCM) at roomtemperature, the glass surfaces were dried and stored at 4° C. untilfurther use.

Example 2 Maleinimido Functionalisation of Poly-Lysine-Modified GlassSurfaces

[0179] 4-maleinimidocaproic acid (Fluka, 63176) was dissolved to 0.3M inDMF. The resultant mixture was activated by adding a half equivalent ofDIC for 15 min at room temperature. Poly-lysine-modified glass surfacespurified with compressed air (Sigma, Poly-Prep™, P₀₄₂₅, 2.5×7.5 cm) wereincubated with the maleinimidocaproic acid anhydride solution thusobtained for three hours at room temperature. In this case 60 μL of thissolution was applied by capillary forces into the gap formed by twomodified glass surfaces located one on top of the other. The glasssurfaces thus treated were then washed five times using respectively 30mL of DMF for 3 minutes each at room temperature. After washing threetimes for three minutes each using respectively 30 mL of DCM at roomtemperature, the glass surfaces were dried and stored at 4° C. untilfurther use.

Example 3 Aldehyde Functionalisation of Aminopropylsilylated GlassSurfaces

[0180] 4-carboxybenzaldehyde (Fluka, 21873) was dissolved to 0.3M inDMF. The resultant mixture was activated by adding a half equivalent ofDIC for 15 min at room temperature. Aminopropylsilylated glass surfacespurified with compressed air (2.5×7.5 cm; Sigma, Silane-Prep™, S4651)were coated with the activated carboxybenzaldehyde solution thusobtained and incubated for three hours at room temperature. The glasssurfaces thus treated were then washed five times using respectively 30mL of DMF for 3 minutes each at room temperature. After washing threetimes for three minutes each using respectively 30 mL of DCM at roomtemperature, the glass surfaces were dried and stored at 4° C. untilfurther use.

Example 4 Ketone Functionalisation of Aminopropylsilylated GlassSurfaces

[0181] Laevulinic acid (Fluka, 21873) was dissolved to 0.3M in DMF. Theresultant mixture was activated by adding a half equivalent of DIC for15 min at room temperature. Aminopropylsilylated glass surfaces purifiedwith compressed air (2.5×7.5 cm; Sigma, Silane-Prep™, S4651) were coatedwith the laevulinic acid anhydride solution thus obtained and incubatedfor three hours at room temperature. The glass surfaces thus treatedwere then washed five times using respectively 30 mL of DML for 3minutes each at room temperature. After washing three times for threeminutes each using respectively 30 mL of DCM at room temperature, theglass surfaces were dried and stored at 4° C. until further use.

Example 5 Bromoacetylation of Aminopropylsilylated glass Surfaces

[0182] Bromoacetic acid was dissolved to 0.4M in DMF. The resultantmixture was activated by adding a half equivalent of DIC for 15 min atroom temperature. Aminopropylsilylated glass surfaces purified withcompressed air (2.5×7.5 cm; Sigma, Silane-Prep™, S4651) were coated withthe bromoacetic acid anhydride solution thus obtained and incubated forthree hours at room temperature. The glass surfaces thus treated werethen washed five times using respectively 30 mL of DMF for 3 minuteseach at room temperature. After washing three times for three minuteseach using respectively 30 mL of DCM at room temperature, the glasssurfaces were dried and stored at 4° C. until further use.

Example 6 4-Bromomethyl Benzoic acid Functionalisation ofAminopropylsilylated Glass Surfaces

[0183] 4-bromomethyl benzoic acid was dissolved to 0.3M in DMF. Theresultant mixture was activated by adding a half equivalent of DIC for15 min at room temperature. Aminopropylsilylated glass surfaces purifiedwith compressed air (2.5×7.5 cm; Sigma, Silane-Prep™, S4651) were coatedwith the 4-bromomethyl benzoic acid anhydride solution thus obtained andincubated for three hours at room temperature. The glass surfaces thustreated were then washed five times using respectively 30 mL of DMF for3 minutes each at room temperature. After washing three times for threeminutes each using respectively 30 mL of DCM at room temperature, theglass surfaces were dried and stored at 4° C. until further use.

Example 7 Phenylthiourea Functionalisation of Aminopropylsilylated GlassSurfaces

[0184] 4-carboxyphenylthiourea (Lancaster, 13047) was dissolved to 0.2Min DMF. The resultant mixture was activated by adding a half equivalentof DIC for 15 min at room temperature. Aminopropylsilylated glasssurfaces purified with compressed air (2.5×7.5 cm; Sigma, Silane-Prep™,S4651) were coated with the activated solution thus obtained andincubated for three hours at room temperature. The glass surfaces thustreated were then washed five times using respectively 30 mL of DMF for3 minutes each at room temperature. After washing three times for threeminutes each using respectively 30 mL of DCM at room temperature, theglass surfaces were dried and stored at 4° C. until further use.

Example 8 Thioamide Functionalisation of Aminopropylsilylated GlassSurfaces

[0185] Succinic acid-mono-thioamide was dissolved to 0.2M in DMF. Theresultant mixture was activated by adding a half equivalent of DIC for15 min at room temperature. Aminopropylsilylated glass surfaces purifiedwith compressed air (2.5×7.5 cm; Sigma, Silane-Prep™ S4651) were coatedwith the modified succinic acid anhydride solution thus obtained andincubated for three hours at room temperature. The glass surfaces thustreated were then washed five times using respectively 30 mL of DMF for3 minutes each at room temperature. After washing three times for threeminutes each using respectively 30 mL of DCM at room temperature, theglass surfaces were dried and stored at 4° C. until further use.

Example 9 Bromoketone Functionalisation of Aminopropylsilylated GlassSurfaces

[0186] 1,4-dibromo-2,3-diketobutane (Aldrich, D3,91609) was dissolved to0.2M in DMF 0.1% triethylamine. Aminopropylsilylated glass surfacespurified with compressed air (2.5×7.5 cm; Sigma, Silane-Prep™ S4651)were coated with solution thus obtained and incubated for seven hours atroom temperature. The glass surfaces thus treated were then washed fivetimes using respectively 30 mL of DMF for 3 minutes each at roomtemperature. After washing three times for three minutes each usingrespectively 30 mL of DCM at room temperature, the glass surfaces weredried and stored at 4° C. until further use.

Example 10 Bromoketone Functionalisation of Thioamide-Modified GlassSurfaces

[0187] The structures shown in this example can be used for simplesurface modification with good yields.

[0188] Aminopropylsilylated glass surfaces reacted with succinic acidmono-thioamide (2.5×7.5 cm; Sigma, Silane-Prep™, S4651) (see Example 8)were coated with a 0.1M 1,4-dibromo-2,3-diketobutane solution (Aldrich,D3,916-9) in ethanol and incubated for three hours at room temperature.The glass surfaces thus treated were then washed five times usingrespectively 30 mL of ethanol for 3 minutes each at room temperature.After washing three times for three minutes each using respectively 30mL of DCM at room temperature, the glass surfaces were dried and storedat 4° C. until further use.

Example 11 Bromoketone Functionalisation of Phenylthiourea-ModifiedGlass Surfaces

[0189] The structures shown in this example can be used for simplesurface modification with good yields.

[0190] Aminopropylsilylated glass surfaces reacted with4-carboxyphenylthiourea (2.5×7.5 cm; Sigma, Silane-Prep™, S4651) werecoated with a 0.1M 1,4-dibromo-2,3-diketobutane solution in ethanol andincubated for three hours at room temperature. The glass surfaces thustreated were then washed five times using respectively 30 mL of ethanolfor 3 minutes each at room temperature. After washing three times forthree minutes each using respectively 30 mL of DCM at room temperature,the glass surfaces were dried and stored at 4° C. until further use.

Example 12 Bromopyroracemic Acid Functionalisation ofAminopropylsilylated Glass Surfaces

[0191] These surface modifications show that even very small structurescan be used to convert an amino-functionalised glass surface to abromoketone-functionalised one. In this case, bromopyroracemic acid isthe smallest possible compound which contains both the carboxyl functionrequired for the amide bond linkage and also the alpha-bromo-ketofunction required for the subsequent immobilisation of the biomolecule.

[0192] Sodium pyruvate was converted with oxalyl chloride into thecorresponding acid chloride. Aminopropylsilylated glass surfacespurified with compressed air (2.5×7.5 cm; Sigma, Silane-Prep™, S4651)were coated with the solution thus obtained and incubated for five hoursat room temperature. The glass surfaces thus treated were then washedfive times using respectively 30 mL of DMF for 3 minutes each at roomtemperature. After washing three times for three minutes each usingrespectively 30 mL of methanol and DCM at room temperature, the glasssurfaces were dried. The pyroracemic acid-modified glass surfaces thusobtained were converted into the bromopyroracemic-acid-modified glasssurfaces by treating for one hour with a solution of 0.1 mL of brominein glacial acetic acid. After washing three times for three minutes eachusing respectively 30 mL of methanol and DCM at room temperature, theglass surfaces were dried and stored at 4° C. until further use.

Example 13 Bromoacetophenone Functionalisation of AminopropylsilylatedGlass Surfaces

[0193] 4-acetylbenzoic acid was dissolved to 0.3M in DMF. The resultantmixture was activated by adding a half equivalent of DIC for 15 min atroom temperature. Aminopropylsilylated glass surfaces purified withcompressed air (2.5×7.5 cm; Sigma, Silane-Prep™, S4651) were coated withthe 4-acetylbenzoic acid anhydride solution thus obtained and incubatedfor three hours at room temperature. The glass surfaces thus treatedwere then washed five times using respectively 30 mL of DMF for 3minutes each at room temperature. After washing three times for threeminutes each using respectively 30 mL of methanol and DCM at roomtemperature, the glass surfaces were dried. The glass surfaces thusmodified were converted into the bromoacetophenone-modified glasssurfaces by treating for one hour with a solution of 0.1 mL of brominein 10 mL of glacial acetic acid. After washing three times for threeminutes each using respectively 30 mL of methanol and DCM at roomtemperature, the glass surfaces were dried and stored at 4° C. untilfurther use.

Example 14 Thiocyanato-acetophenone Functionalisation ofAminopropylsilylated Glass Surfaces

[0194] Bromoacetophenone-modified aminopropylsilylated glass surfaces(2.5×7.5 cm; Sigma, Silane-Prep™ S4651) (see Example 13) were coatedwith a 0.1M solution of sodium thiocyanate in ethanol and incubated forfive hours at 50° C. The glass surfaces thus treated were then washedfive times using respectively 30 mL of ethanol for 3 minutes each atroom temperature. After washing three times for three minutes each usingrespectively 30 mL of DCM at room temperature, the glass surfaces weredried and stored at 4° C. until further use.

Example 15 Immobilisation of Cysteine-Containing Peptides onMaleinimido-Functionalised Glass Surfaces

[0195] a) The peptides used for the immobilisation were synthesised bystandard methods of Fmoc-based chemistry on the solid phase asC-terminal peptide amides. In this case, correspondingly protected Fmocamino acids were activated with one equivalent HBTU and threeequivalents diisopropylethylamine in DMF and coupled to Rink amide MBHAresin in DMF. Cleaving of the Fmoc protective group was carried outusing 20% piperidine in DMF for 30 min at room temperature. Cleaving ofthe permanent protective groups (tBu for serine, threonine, tyrosine,glutaminic acid and asparaginic acid; Boc for lysine; trityl forasparagine, glutamine, cysteine, histidine and Pbf for arginine) and thesimultaneous detachment from the polymer was carried out by treatmentfor two hours using 97% trifluoroacetic acid at room temperature. Theresultant mixture was filtered and the filtrate was precipitated byadding tert-butyl methyl ether. The precipitate was separated andpurified using HPLC on RP18 material using acetonitrile/water mixtures(0.1% trifluoroacetic acid). The fractions containing the desiredproduct were lyophilised and stored at −20° C. until further use.

[0196] b) The HPLC-purified cysteine-containing peptides were dissolvedin 200 mM sodium phosphate buffer pH 7.5 (final peptide concentration 10mM). Then, respectively 1 μL of this solution was spotted onto themaleinimido-functionalised glass surfaces (see Example 1) at roomtemperature using an Eppendorf pipette and this was incubated for onehour at room temperature in an almost water-saturated atmosphere. Afterwashing three times at room temperature using 100 mL of distilled watereach time, the modified glass surfaces were incubated with 30 mL of a300 mM solution of mercaptoethanol in 200 mM sodium phosphate buffer pH7.5 to deactivate the residual maleinimido functions. The glass surfaceswere then washed five times for 3 minutes at a time using respectively50 mL of water at room temperature and then washed twice for 3 minutesat a time using respectively 50 mL of methanol at room temperature. Theglass surfaces thus treated were dried and stored at 4° C. until furtheruse.

Example 16 Immobilisation of Cysteine-Containing Peptides onBromoacetylated Glass Surfaces

[0197] The HPLC-purified cysteine-containing peptides were dissolved in200 mM sodium phosphate buffer pH 6.5 (final peptide concentration 5mM). Then, respectively 1 μL of this solution was spotted onto thefunctionalised glass surfaces (see Example 5) at room temperature usingan Eppendorf pipette and this was incubated for one hour at roomtemperature in an almost water-saturated atmosphere. After washing threetimes at room temperature using 100 mL of distilled water each time, themodified glass surfaces were incubated with 30 mL of a 300 mM solutionof mercaptoethanol in 200 mM sodium phosphate buffer pH 7.5 todeactivate the residual maleinimido functions. The glass surfaces werethen washed five times for 3 minutes at a time using respectively 50 mLof water at room temperature and then washed twice for 3 minutes at atime using respectively 50 mL of methanol at room temperature. The glasssurfaces thus treated were dried and stored at 4° C. until further use.

Example 17 Immobilisation of Cysteine-Containing Peptides onAldehyde-Functionalised Glass Surfaces

[0198] The purified cysteine-containing peptides were dissolved in 200mM sodium phosphate buffer pH 5.5 (final peptide concentration 5 mM)containing 200 mM tris-carboxyethyl phosphine. Then, respectively 1 μLof this solution was spotted onto the aldehyde-functionalised glasssurfaces (see Example 3) at room temperature using an Eppendorf pipetteand this was incubated for four hours at room temperature in an almostwater-saturated atmosphere. After washing three times at roomtemperature using 100 mL of distilled water each time, the modifiedglass surfaces were incubated with 30 mL of a 40% aqueous solution ofhydroxylamine for 30 minutes at room temperature to deactivate theresidual aldehyde functions. The glass surfaces were then washed fivetimes for 3 minutes at a time using respectively 50 mL of water at roomtemperature and then washed twice for 0.3 minutes at a time usingrespectively 50 mL of methanol at room temperature. The glass surfacesthus treated were dried and stored at 4° C. until further use.

Example 18 Immobilisation of Cysteine-Containing Peptides onMaleinimido-Functionalised Glass Surfaces

[0199] a) The peptides used for the immobilisation (43serine/threonine-containing peptides and the corresponding 43 controlpeptides) were synthesised by standard SPOT methods synthesis (R. Frank,Tetrahedron, 48, 1992, pp. 9217-9232; A. Kramer and J.Schneider-Mergener, Methods in Molecular Biology, vol. 87: CombinatorialPeptide Library Protocols, p. 25-39, edited by: S. Cabilly; HumanaPress. Inc. Totowa, N.J.) on cellulose as C-terminal peptide amides. Inthis case, correspondingly protected Fmoc amino acid pentafluorophenylesters were dissolved in DMF and 1 μL at a time was spotted on. Thecoupling reaction took place twice for 25 min at a time at roomtemperature. Cleaving of the Fmoc protective group was carried out using20% piperidine in DMF for 20 min at room temperature. Cleaving of thepermanent protective groups (tBu for serine, threonine, tyrosine,glutaminic acid and asparaginic acid; Boc for lysine; trityl forasparagine, glutamine, cysteine, histidine and Pbf for arginine) wascarried out by treatment for two hours using 97% trifluoroacetic acid atroom temperature. The cellulose-bound peptides were then washed withDCM, MeOH and diethylether and dried in vacuum. The peptides werecleaved from the cellulose using ammonia gas for 24 hours at roomtemperature. The spots with the physically adsorbed peptides werepunched out and transferred to 96-well microtiter plates. Afterdetaching the peptides using 200 μL of 20% methanol in each case underultrasound conditions, the samples were filtered, transferred to384-well microtiter plates, lyophilised and stored at −20° C. untilfurther use. The following list gives an overview of the synthesisedpeptide sequences and at the same time allows the peptide numbers inFIGS. 8 and 9 to be assigned to the corresponding sequences(BA1=β-alanine). 1Cys-BAl-Lys-Lys-Ala-Leu-Arg-Arg-Gln-Glu-Thr-Val-Asp-Ala-Leu-NH₂ 2Cys-BAl-Lys-Lys-Ala-Leu-Arg-Arg-Gln-Glu-Ala-Val-Asp-Ala-Leu-NH₂ 3Cys-BAl-Ala-Lys-Arg-Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala-NH₂ 4Cys-BAl-Ala-Lys-Arg-Arg-Arg-Leu-Ala-Ala-Leu-Arg-Ala-NH₂ 5Cys-BAl-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ser-Ile-NH₂ 6Cys-BAl-Gly-Arg-Ala-Gly-Arg-Arg-Asn-Ala-Ile-NH₂ 7Cys-BAl-Asp-Asp-Asp-Glu-Glu-Ser-Ile-Thr-Arg-Arg-NH₂ 8Cys-BAl-Asp-Asp-Asp-Glu-Glu-Ala-Ile-Ala-Arg-Arg-NH₂ 9Cys-BAl-Glu-Arg-Ser-Pro-Ser-Pro-Ser-Phe-Arg-NH₂ 10Cys-BAl-Glu-Arg-Ala-Pro-Ala-Pro-Ala-Phe-Arg-NH₂ 11Cys-BAl-Gly-Arg-Pro-Arg-Thr-Ser-Ser-Phe-Ala-Glu-Gly-NH₂ 12Cys-BAl-Gly-Arg-Pro-Arg-Ala-Ala-Ala-Phe-Ala-Glu-Gly-NH₂ 13Cys-BAl-Lys-Lys-Lys-Ala-Leu-Ser-Arg-Gln-Leu-Ser-Val-Ala-Ala-NH₂ 14Cys-BAl-Lys-Lys-Lys-Ala-Leu-Ala-Arg-Gln-Leu-Ala-Val-Ala-Ala-NH₂ 15Cys-BAl-Lys-Lys-Leu-Asn-Arg-Thr-Leu-Ser-Val-Ala-NH₂ 16Cys-BAl-Lys-Lys-Leu-Asn-Arg-Ala-Leu-Ala-Val-Ala-NH₂ 17Cys-BAl-Lys-Arg-Gln-Gln-Ser-Phe-Asp-Leu-Phe-NH₂ 18Cys-BAl-Lys-Arg-Gln-Gln-Ala-Phe-Asp-Leu-Phe-NH₂ 19Cys-BAl-Lys-Arg-Arg-Glu-Ile-Leu-Ser-Arg-Arg-Pro-Ser-Tyr-Arg-NH₂ 20Cys-BAl-Lys-Arg-Arg-Glu-Ile-Leu-Ala-Arg-Arg-Pro-Ala-Phe-Arg-NH₂ 21Cys-BAl-Leu-Arg-Ala-Pro-Ser-Trp-Ile-Asp-Thr-NH₂ 22Cys-BAl-Leu-Arg-Ala-Pro-Ala-Trp-Ile-Asp-Ala-NH₂ 23Cys-BAl-Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala-Lys-Lys-NH₂ 24Cys-BAl-Pro-Leu-Ala-Arg-Ala-Leu-Ala-Val-Ala-Ala-Lys-Lys-NH₂ 25Cys-BAl-Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ser-Ser-NH₂ 26Cys-BAl-Pro-Leu-Ala-Arg-Ala-Leu-Ala-Val-Ala-Ala-NH₂ 27Cys-BAl-Gln-Lys-Arg-Pro-Ser-Gln-Arg-Ser-Lys-Tyr-Leu-NH₂ 28Cys-BAl-Gln-Lys-Arg-Pro-Ala-Gln-Arg-Ala-Lys-Phe-Leu-NH₂ 29Cys-BAl-Arg-Lys-Ile-Ser-Ala-Ser-Glu-Phe-NH₂ 30Cys-BAl-Arg-Lys-Ile-Ala-Ala-Ala-Glu-Phe-NH₂ 31Pro-Lys-Thr-Pro-Lys-Lys-Ala-Lys-Lys-Leu-BAl-Cys-NH₂ 32Pro-Lys-Ala-Pro-Lys-Lys-Ala-Lys-Lys-Leu-BAl-Cys-NH₂ 33Cys-BAl-Arg-Pro-Arg-Ala-Ala-Thr-Phe-NH₂ 34Cys-BAl-Arg-Pro-Arg-Ala-Ala-Ala-Phe-NH₂ 35Cys-BAl-Arg-Arg-Arg-Ala-Pro-Leu-Ser-Pro-NH₂ 36Cys-BAl-Arg-Arg-Arg-Ala-Pro-Leu-Ala-Pro-NH₂ 37Cys-BAl-Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu-NH₂ 38Cys-BAl-Arg-Arg-Arg-Glu-Glu-Glu-Ala-Glu-Glu-Glu-NH₂ 39Cys-BAl-Met-His-Arg-Gln-Glu-Thr-Val-Asp-Cys-Leu-Lys-NH₂ 40Cys-BAl-Met-His-Arg-Gln-Glu-Ala-Val-Asp-Cys-Leu-Lys-NH₂ 41Cys-BAl-Lys-Lys-Arg-Phe-Ser-Phe-Lys-Lys-Ser-Phe-Lys-Leu-NH₂ 42Cys-BAl-Lys-Lys-Arg-Phe-Ala-Phe-Lys-Lys-Ala-Phe-Lys-Leu-NH₂ 43Cys-BAl-Pro-Lys-Asp-Pro-Ser-Gln-Arg-Arg-Arg-NH₂ 44Cys-BAl-Pro-Lys-Asp-Pro-Ala-Gln-Arg-Arg-Arg-NH₂ 45Cys-BAl-Ile-Ala-Ala-Asp-Ser-Glu-Ala-Glu-Gln-NH₂ 46Cys-BAl-Ile-Ala-Ala-Asp-Ala-Glu-Ala-Glu-Gln-NH₂ 47Cys-BAl-Ser-Pro-Ala-Leu-Thr-Gly-Asp-Glu-Ala-NH₂ 48Cys-BAl-Ala-Pro-Ala-Leu-Ala-Gly-Asp-Glu-Ala-NH₂ 49Cys-BAl-Gly-Arg-Ile-Leu-Thr-Leu-Pro-Arg-Ser-NH₂ 50Cys-BAl-Gly-Arg-Ile-Leu-Ala-Leu-Pro-Arg-Ala-NH₂ 51Cys-BAl-Met-Gly-Glu-Ala-Ser-Gly-Cys-Gln-Leu-NH₂ 52Cys-BAl-Met-Gly-Glu-Ala-Ala-Gly-Cys-Gln-Leu-NH₂ 53Cys-BAl-Glu-Glu-Thr-Pro-Tyr-Ser-Tyr-Pro-Thr-NH₂ 54Cys-BAl-Glu-Glu-Ala-Pro-Phe-Ser-Phe-Pro-Ala-NH₂ 55Cys-BAl-Gly-Asn-Ths-Thr-Tyr-Gln-Glu-Ile-Ala-NH₂ 56Cys-BAl-Gly-Asn-His-Ala-Phe-Gln-Glu-Ile-Ala-NH₂ 57Leu-Arg-Ser-Pro-Ser-Trp-Glu-Pro-Phe-BAl-Cys-NH₂ 58Leu-Arg-Ala-Pro-Ala-Trp-Glu-Pro-Phe-BAl-Cys-NH₂ 59Ser-Ser-Pro-Val-Tyr-Gln-Asp-Ala-Val-BAl-Cys-NH₂ 60Ala-Ala-Pro-Val-The-Gln-Asp-Ala-Val-BAl-Cys-NH₂ 61Cys-BAl-Ser-Arg-Thr-Leu-Ser-Val-Ser-Ser-Leu-NH₂ 62Cys-BAl-Ala-Arg-Ala-Leu-Ala-Val-Ala-Ala-Leu-NH₂ 63Leu-Ser-Val-Ser-Ser-Leu-Pro-Gly-Leu-BAl-Cys-NH₂ 64Leu-Ser-Val-Ala-Ala-Leu-Pro-Gly-Leu-BAl-Cys-NH₂ 65Cys-BAl-Val-Thr-Pro-Arg-Thr-Pro-Pro-Pro-Ser-NH₂ 66Cys-BAl-Val-Ala-Pro-Arg-Ala-Pro-Pro-Pro-Ala-NH₂ 67Cys-BAl-Arg-Phe-Ala-Arg-Lys-Gly-Ser-Leu-Arg-Gln-Lys-Ans-Val-NH₂ 68Cys-BAl-Arg-Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln-Lys-Ans-Val-NH₂ 69Cys-BAl-Pro-Arg-Pro-Ala-Ser-Val-Pro-Pro-Ser-NH₂ 70Cys-BAl-Pro-Arg-Pro-Ala-Ser-Ala-Val-Pro-Pro-Ala-NH₂ 71Cys-BAl-Arg-Glu-Ala-Arg-Ser-Arg-Ala-Ser-Thr-NH₂ 72Cys-BAl-Arg-Glu-Ala-Arg-Ala-Arg-Ala-Ala-Ala-NH₂ 73Gln-Ser-Tyr-Ser-Ser-Ser-Gln-Arg-Val-BAl-Cys-NH₂ 74Gln-Ser-Tyr-Ala-Ala-Ala-Gln-Arg-Val-BAl-Cys-NH₂ 75Cys-BAl-Gly-Gly-Gly-Thr-Ser-Pro-Val-Phe-Pro-NH₂ 76Cys-BAl-Gly-Gly-Gly-Ala-Ala-Pro-Val-Phe-Pro-NH₂ 77Leu-Tyr-Ser-Ser-Ser-Pro-Gly-Gly-Ala-BAl-Cys-NH₂ 78Leu-Tyr-Ala-Ala-Ala-Pro-Gly-Gly-Ala-BAl-Cys-NH₂ 79Cys-BAl-Asp-Leu-Pro-Leu-Ser-Pro-Ser-Ala-Phe-NH₂ 80Cys-BAl-Asp-Leu-Pro-Leu-Ala-Pro-Ala-Ala-Phe-NH₂ 81Cys-BAl-Thr-Thr-Pro-Leu-Ser-Pro-Thr-Arg-Leu-NH₂ 82Cys-BAl-Ala-Ala-Pro-Leu-Ala-Pro-Ala-Arg-Leu-NH₂ 83Arg-Arg-Ile-Ser-Lys-Asp-Asn-Pro-Asp-Tyr-Gln-Gln-Asp-BAl-Cys-NH₂ 84Arg-Arg-Ile-Ala-Lys-Asp-Asn-Pro-Asp-Tyr-Gln-Gln-Asp-BAl-Cys-NH₂ 85Cys-BAl-Leu-Arg-Arg-Ala-Ser-Leu-Gly-NH₂ 86Cys-BAl-Leu-Arg-Arg-Ala-Ala-Leu-Gly-NH₂

[0200] b) The cysteine-containing peptides in the microtiter plates wereapplied to the maleinimido-functionalised glass surfaces (see Example 1)by using a NanoPlotter from Gesim. The spot-to-spot distance was 1 mm.0.8 nL of a peptide solution in 100 mM PBS buffer pH 7.8 containing 20%glycerin was applied per spot and this assembly was incubated for fourhours at room temperature. After washing three times at room temperatureusing 100 mL of distilled water each time, the modified glass surfaceswere incubated with 30 mL of a 300 mM solution of mercaptoethanol in 200mM phosphate buffer pH 7.5 to deactivate the residual maleinimidofunctions. The glass surfaces were then washed five times for 3 minutesat a time using respectively 50 mL of water at room temperature and thenwashed twice for 3 minutes at a time using respectively 50 mL ofmethanol at room temperature. The glass surfaces thus treated were driedand stored at 4° C. until further use.

Example 19 Inmobilisation of Anthraniloyl Peptides onAldehyde-Functionalised Glass Surfaces

[0201] a) The peptides used for the immobilisation (in each case 13-merpeptides representing the total primary structure of the proteins MBP,casein and histon H1) were synthesised by standard SPOT methodssynthesis (R. Frank, Tetrahedron, 48, 1992, pp. 9217-9232; A. Kramer andJ. Schneider-Mergener, Methods in Molecular Biology, vol. 87:Combinatorial Peptide Library Protocols, p. 25-39, edited by: S.Cabilly; Humana Press. Inc. Totowa, N.J.) on cellulose as C-terminalpeptide amides. In this case, correspondingly protected Fmoc amino acidpentafluorophenyl esters were dissolved in DMF and 1 μL at a time wasspotted on. The coupling reaction took place twice for 25 min at a timeat room temperature. Cleaving of the Fmoc protective group was carriedout using 20% piperidine in DMF for 20 min at room temperature. Afterthe last Fmoc cleaving the N-termini of the cellulose-bound peptideswere converted into the corresponding 2-aminobenzoylated derivatives byincubation for five hours at 50° C. using a saturated solution of Isaturacid in DMF. Cleaving of the permanent protective groups (tBu forserine, threonine, tyrosine, glutaminic acid and asparaginic acid; Bocfor lysine; trityl for asparagine, glutamine, cysteine, histidine andPbf for arginine) was carried out by treatment for two hours using 97%trifluoroacetic acid at room temperature. The cellulose-bound peptideswere then washed with DCM, MeOH and diethylether and dried in vacuum.The peptides were cleaved from the cellulose using ammonia gas for 24hours at room temperature. The spots with the physically adsorbedpeptides were punched out and transferred to 96-well microtiter plates.After detaching the peptides using 200 μL of 20% methanol in each caseunder ultrasound conditions, the samples were filtered, transferred to384-well microtiter plates, lyophilised and stored at −20° C. untilfurther use.

[0202] b) The anthraniloyl peptides in the microtiter plates weredissolved in 200 mM sodium phosphate buffer pH 6.0 (final peptideconcentration 0.5 mM) containing 15% DMSO. Then 0.01 μL of this solutionat a time was applied to the aldehyde-modified glass surfaces (seeExample 3) at room temperature using a NanoPlotter from Gesim and thiswas incubated for four hours at room temperature. After washing threetimes at room temperature using 100 mL of distilled water each time, themodified glass surfaces were incubated with 30 mL of 40% aqueoussolution of hydroxylamine for 30 minutes at room temperature todeactivate the residual aldehyde functions. The glass surfaces were thenwashed five times for 3 minutes at a time using respectively 50 mL ofwater at room temperature and then washed twice for 3 minutes at a timeusing respectively 50 mL of methanol at room temperature. The glasssurfaces thus treated were dried and stored at 4° C. until further use.

Example 20 Immobilisation of Amino-Oxyacetic-Acid Containing Peptides onAldehyde-Functionalised Glass Surfaces

[0203] a) The peptides used for the immobilisation (in each case 13-merpeptides representing the total primary structure of the proteins MBP,casein and histon H1) were synthesised by standard SPOT methodssynthesis (R. Frank, Tetrahedron, 48, 1992, pp. 9217-9232; A. Kramer andJ. Schneider-Mergener, Methods in Molecular Biology, vol. 87:Combinatorial Peptide Library Protocols, p. 25-39, edited by: S.Cabilly; Humana Press. Inc. Totowa, N.J.) on cellulose as C-terminalpeptide amides. In this case, correspondingly protected Fmoc amino acidpentafluorophenyl esters were dissolved in DMF and 1 μL at a time wasspotted on. The coupling reaction took place twice for 25 min at a timeat room temperature. Cleaving of the Fmoc protective group was carriedout using 20% piperidine in DMF for 20 min at room temperature. TheN-terminus was acylated using Boc-amino-oxy-acetic acid. For thispurpose this was activated in DMF using 1 equivalent HOAT/DIC. In eachcase 1 μL of this mixture was spotted onto each cellulose-bound peptideand left for 30 min at room temperature. Cleaving of the permanentprotective groups (tBu for serine, threonine, tyrosine, glutaminic acidand asparaginic acid; Boc for lysine; trityl for asparagine, glutamine,cysteine, histidine and Pbf for arginine) was carried out by treatmentfor two hours using 97% trifluoroacetic acid at room temperature. Thecellulose-bound peptides were then washed with DCM, MeOH anddiethylether and dried in vacuum. The peptides were cleaved from thecellulose using ammonia gas for 24 hours at room temperature. The spotswith the physically adsorbed peptides were punched out and transferredto 96-well microtiter plates. After detaching the peptides using 200 μLof 20% methanol in each case under ultrasound conditions, the sampleswere filtered, transferred to 384-well microtiter plates, lyophilisedand stored at −20° C. until further use.

[0204] b) The amino-oxy-acetic acid-containing peptides in themicrotiter plates were dissolved in DMSO. Then 1 nL of this solution ata time was applied to the aldehyde-functionalised glass surfaces (seeExample 3) at room temperature using a NanoPlotter from Gesim and thiswas incubated for four hours at room temperature. In this case, thespot-to-spot distance was 1.5 mm. After washing three times at roomtemperature using 100 mL of distilled water each time, the modifiedglass surfaces were incubated with 30 mL of a 40% aqueous solution ofhydroxylamine for 30 minutes at room temperature to deactivate theresidual aldehyde functions. The glass surfaces were then washed fivetimes for 3 minutes at a time using respectively 50 mL of water at roomtemperature and then washed twice for 3 minutes at a time usingrespectively 50 mL of methanol at room temperature. The glass surfacesthus treated were dried and stored at 4° C. until further use.

Example 21 Immobilisation of Amino-Oxyacetic-Acid Containing Peptides onBromo-Acetylated Glass Surfaces

[0205] a) The peptides used for the immobilisation (in each case 13-merpeptides representing the total primary structure of the proteins MBP,casein and histon H1) were synthesised by standard SPOT methodssynthesis (R. Frank, Tetrahedron, 48, 1992, pp. 9217-9232; A. Kramer andJ. Schneider-Mergener, Methods in Molecular Biology, vol. 87:Combinatorial Peptide Library Protocols, p. 25-39, edited by: S.Cabilly; Humana Press. Inc. Totowa, N.J.) on cellulose as C-terminalpeptide amides. In this case, correspondingly protected Fmoc amino acidpentafluorophenyl esters were dissolved in DMF and 1 μL at a time wasspotted on. The coupling reaction took place twice for 25 min at a timeat room temperature. Cleaving of the Fmoc protective group was carriedout using 20% piperidine in DMF for 20 min at room temperature. TheN-terminus was acylated using Boc-amino-oxy-acetic acid. For thispurpose this was activated in DMF using 1 equivalent HOAT/DIC. In eachcase 1 μL of this mixture was spotted onto each cellulose-bound peptideand left for 30 min at room temperature. Cleaving of the permanentprotective groups (tBu for serine, threonine, tyrosine, glutaminic acidand asparaginic acid; Boc for lysine; trityl for asparagine, glutamine,cysteine, histidine and Pbf for arginine) was carried out by treatmentfor two hours using 97% trifluoroacetic acid at room temperature. Thecellulose-bound peptides were then washed with DCM, MeOH anddiethylether and dried in vacuum. The peptides were cleaved from thecellulose using ammonia gas for 24 hours at room temperature. The spotswith the physically adsorbed peptides were punched out and transferredto 96-well microtiter plates. After detaching the peptides using 200 μLof 20% methanol in each case under ultrasound conditions, the sampleswere filtered, transferred to 384-well microtiter plates, lyophilisedand stored at −20° C. until further use.

[0206] b) The amino-oxy-acetic acid-containing peptides in themicrotiter plates were dissolved in 200 mM sodium phosphate buffer pH6.0 (final peptide concentration 0.5 mM) containing 25 vol. % glycerin.Then 0.01 μL of this solution at a time was applied to thebromo-acetylated, amino-functionalised glass surfaces (see Example 5) atroom temperature using a NanoPlotter from Gesim and this was incubatedfor four hours at room temperature. After washing three times at roomtemperature using 100 mL of distilled water each time, the modifiedglass surfaces were incubated with 30 mL of a 300 mM solution ofmercaptoethanol in 200 mM sodium phosphate buffer pH 7.5 to deactivatethe residual bromo-acetyl functions. The glass surfaces were then washedfive times for 3 minutes at a time using respectively 50 mL of water atroom temperature and then washed twice for 3 minutes at a time usingrespectively 50 mL of methanol at room temperature. The glass surfacesthus treated were dried and stored at 4° C. until further use.

Example 22 Immobilisation of Amino-Oxyacetic-Acid Containing Peptides onAldehyde-Functionalised Glass Surfaces

[0207] a) The peptide used for the immobilisation,Dpr(Aoa)-Leu-Arg-Arg-Ala-Ser-Leu-Gly-NH₂ was synthesised by standardmethods of Fmoc-based chemistry on the solid phase as C-terminal peptideamide. In this case, correspondingly protected Fmoc amino acids wereactivated with one equivalent HBTU and three equivalentsdiisopropylethyl amine in DMF and coupled to Rink amide MBHA resin inDMF. Cleaving of the Fmoc protective group was carried out using 20%piperidine in DMF for 30 min at room temperature. Cleaving of thepermanent protective groups (tBu for serine, Boc for the amino-oxyfunction and Pbf for arginine) and simultaneous detachment from thepolymer was carried out by treatment for two hours using 97%trifluoroacetic acid at room temperature. The resulting mixture wasfiltered and the filtrate was precipitated by adding tert-butyl methylether. The precipitate was separated and purified by means of HPLC onRP18 material using acetonitrile/water mixtures (0.1% trifluoroaceticacid). The fractions containing the desired product were lyophilised andstored at −20° C. until further use.

[0208] b) The peptide Dpr(Aoa)-Leu-Arg-Arg-Ala-Ser-Leu-Gly-NH₂ wasdissolved in 200 mM acetate buffer pH 4.0 (final peptide concentration0.5 mM) containing 25 vol. % tert-butanol. Then 5 nL of this solution ata time was applied to the aldehyde-functionalised glass surfaces(Telechem/ArrayIt, CSS-25 glass support) in an assembly of 20 rows and48 gaps (a total of 960 spots) at room temperature using a NanoPlotterfrom Gesim. In this case, the spot-to-spot distance was 1 mm. The glasssurfaces thus treated were then incubated for four hours at roomtemperature. After washing three times at room temperature using 100 mLof distilled water each time, the modified glass surfaces were incubatedwith 30 mL of a 40% aqueous solution of hydroxylamine 30 min at roomtemperature to deactivate the residual aldehyde functions. The glasssurfaces were then washed five times for 3 minutes at a time usingrespectively 50 mL of water at room temperature and then washed twicefor 3 minutes at a time using respectively 50 mL of methanol at roomtemperature. The glass surfaces thus treated were dried and stored at 4°C. until further use.

Example 23 Immobilisation of Cysteine-Containing Peptides on4-Bromomethylbenzoylated Glass Surfaces

[0209] The HPLC-purified peptideCys-βAla-Leu-Arg-Arg-Ala-Ser-Leu-Gly-NH₂ was dissolved in 200 mM sodiumphosphate buffer pH 6.5 (final peptide concentration 2 mM) containing 20vol. % glycerin. Then 2 nL of this solution at a time was applied to thebromomethylbenzoic-acid-functionalised glass surfaces (see Example 6) inan assembly of 50 rows and 120 gaps (a total of 6000 spots) at roomtemperature using a NanoPlotter from Gesim. In this case, thespot-to-spot distance was 0.4 mm. The glass surfaces thus treated werethen incubated for five hours at room temperature. After washing threetimes at room temperature using 100 mL of distilled water each time, themodified glass surfaces were incubated with 30 mL of a 300 mM solutionof mercaptoethanol in 200 mM sodium phosphate buffer pH 7.5 todeactivate the residual bromomethyl phenyl functions. The glass surfaceswere then washed five times for 3 minutes at a time using respectively50 mL of water at room temperature and then washed twice for 3 minutesat a time using respectively 50 mL of methanol at room temperature. Theglass surfaces thus treated were dried and stored at 4° C. until furtheruse.

Example 24 Immobilisation of Thioamide-Containing Peptides on1-bromo-2,3-diketo-butane-functionalised Glass Surfaces

[0210] a) The peptide used for the immobilisation,Leu-Arg-Arg-Ala-Ser-Leu-Gly-thioamide was synthesised by standardmethods of Fmoc-based chemistry on the solid phase as C-terminal peptideamide. Fmoc-Gly-OH bound to Rink amide MBHA resin was boiled underreflux for 3 hours using Lawessons reagent. The resin was then washedwith THF and DCM, agitated for 1 hour with DMF and then washed with DMF,DCM and methanol. After removing the Fmoc protective group using 50%Morpholine in DMF (40 min), the correspondingly protected Fmoc aminoacids were activated with one equivalent HBTU and three equivalentsdiisopropylethyl amine in DMF and coupled. Cleaving of the Fmocprotective group was carried out using 20% piperidine in DMF for 30 minat room temperature. Cleaving of the permanent protective groups (tBufor serine, Pbf for arginine) and simultaneous detachment from thepolymer was carried out by treatment for two hours using 97%trifluoroacetic acid at room temperature. The resulting mixture wasfiltered and the filtrate was precipitated by adding tert-butyl methylether. The precipitate was separated and purified by means of HPLC onRP18 material using acetonitrile/water mixtures (0.1% trifluoroaceticacid). The fractions containing the desired product were lyophilised andstored at −20° C. until further use.

[0211] b) The peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly-thioamide wasdissolved in 200 mM sodium phosphate buffer pH 5.5 (final concentration1 mM) containing 50 vol. % glycerin. Then 1 nL of this solution at atime was applied to the 1-bromo-2,3-diketo-butane-functionalised glasssurfaces (see Example 10) in an assembly of 70 rows and 168 gaps (atotal of 11760 spots) at room temperature using a NanoPlotter fromGesim. In this case, the spot-to-spot distance was 0.3 mm. The glasssurfaces thus treated were then microwave-treated for 2 min and thenincubated for three hours at room temperature. After washing three timesat room temperature using 100 mL of distilled water each time, themodified glass surfaces were incubated with 30 mL of a 3% aqueoussolution of thioacetamide for 30 min at room temperature to deactivatethe residual α-bromo-ketone functions. The glass surfaces were thenwashed five times for 3 minutes at a time using respectively 50 mL ofwater at room temperature and then washed twice for 3 minutes at a timeusing respectively 50 mL of methanol at room temperature. The glasssurfaces thus treated were dried and stored at 4° C. until further use.

Example 25 Analysis of Kinase-Mediated Peptide Modifications on ModifiedGlass Surfaces (see FIG. 4)

[0212] A glass surface (maleinimido-functionalised glass surface, seeExample 1) modified with the peptideCys-βAla-Leu-Arg-Arg-Ala-Ser-Leu-Gly-NH₂ (both as the raw peptide and asa peptide purified by means of prep. HPLC) and the control peptideCys-βAla-Leu-Arg-Arg-Ala-Ala-Leu-Gly-NH₂ was incubated with 10 mL of 100μM ATP in kinase buffer (50 mM tris-HCl, 150 mM NaCl, 30 mM MgCl₂, 4 mMDTT, 2 mM EGTA, pH 7.5) for 10 minutes at room temperature. Then 1 μL ofa mixture of protein kinase A (Sigma, P26452, U/mL), 100 μM/mL ATP and100 μCi/mL γ-³²P-ATP (Amersham, 9.25 rnBq/250 μCi/25 μL, activity >5000Ci/mmol) in kinase buffer (50 mM tris-HCl, 150 mM NaCl, 30 mM MgCl₂, 4mM DTT, 2 mM EGTA, pH 7.5) was spotted onto the peptide-modified glasssurfaces and incubated for 30 min at room temperature in an almostwater-saturated atmosphere. In order to reduce the background caused bythe non-specific binding of ATP or kinase molecules to the glasssurfaces, the modified glass surfaces were washed as follows:

[0213] three times for 3 minutes at room temperature using washingbuffer (1% SDS and 1% Tween20 in 50 mM TRIS buffer, pH 7.5, 200 mM NaCl)

[0214] twice for 3 minutes at room temperature using 1M NaCl solution

[0215] twice for 3 minutes at room temperature using distilled water

[0216] twice for 3 minutes at room temperature using 80% formic acid inethanol

[0217] twice for 3 minutes at room temperature using distilled water

[0218] twice for 5 minutes at 50° C. using a solution containing 6Murea, 2M thiourea and 1% SDS.

[0219] three times for 3 minutes at room temperature using distilledwater

[0220] three times for 3 minutes at room temperature using methanol

[0221] After drying the glass surface the quantity of radioactivephosphate incorporated in the glass-surface-bound peptides wasdetermined using a PhosphorImager system (FLA-3000, FUJIFILM) (see FIG.4).

Example 26 Analysis of Kinase-Mediated Peptide Modifications on ModifiedGlass Surfaces (see FIG. 5)

[0222] A glass surface (maleinimido-functionalised glass surface, seeExample 1) modified with the peptideCys-βAla-Leu-Arg-Arg-Ala-Ser-Leu-Gly-NH₂ and the control peptideCys-βAla-Leu-Arg-Arg-Ala-Ala-Leu-Gly-NH₂ was incubated with 10 mL of 100μM ATP in kinase buffer (50 mM tris-HCl, 150 mM NaCl, 30 mM MgCl₂, 4 mMDTT, 2 mM EGTA, pH 7.5) for 10 minutes at room temperature. The glasssurface was dried and a cover glass was then placed on thepeptide-modified glass surface. Then 20 μL of a mixture of proteinkinase A (Sigma, P26452, U/mL), 100 μM/mL ATP and 100 μCi/mL γ-³²P-ATP(Amersham, 9.25 mBq/250 μCi/25 μL, activity >5000 Ci/mmol) in kinasebuffer (50 mM tris-HCl, 150 mM NaCl, 30 mM MgCl₂, 4 mM DTT, 2 mM EGTA,pH 7.5) was applied by capillary forces into the gap formed by the coverglass lying on the modified glass surface. Incubation was then carriedout for 30 min at room temperature in an almost water-saturatedatmosphere. In order to reduce the background caused by the non-specificbinding of ATP or kinase molecules to the glass surfaces, the modifiedglass surfaces were washed as follows:

[0223] three times for 3 minutes at room temperature using washingbuffer (1% SDS and 1% Tween20 in 50 mM TRIS buffer, pH 7.5, 200 mM NaCl)

[0224] twice for 3 minutes at room temperature using 1M NaCl solution

[0225] twice for 3 minutes at room temperature using distilled water

[0226] twice for 3 minutes at room temperature using 80% formic acid inethanol

[0227] twice for 3 minutes at room temperature using distilled water

[0228] twice for 5 minutes at 50° C. using a solution containing 6Murea, 2M thiourea and 1% SDS

[0229] three times for 3 minutes at room temperature using distilledwater

[0230] three times for 3 minutes at room temperature using methanol

[0231] After drying the glass surface the quantity of radioactivephosphate incorporated in the glass-surface-bound peptides wasdetermined using a PhosphorImager system (FLA-3000, FUJIFILM) (see FIG.5).

Example 27 Analysis of Kinase-Mediated Peptide Modifications on ModifiedGlass Surfaces (see FIG. 7)

[0232] A glass surface modified with the peptideDpr(Aoa)-Leu-Arg-Arg-Ala-Ser-Leu-Gly-NH₂ (see Example 22) was incubatedwith 10 mL of 100 μM ATP in kinase buffer (50 mM tris-HCl, 150 mM NaCl,30 mM MgCl₂, 4 mM DTT, 2 mM EGTA, pH 7.5) for 10 minutes at roomtemperature. The glass surface was dried and a second unmodified glasssurface of the same dimensions was then placed on the peptide-modifiedglass surface. Then 50 μL of a mixture of protein kinase A (Sigma,P26452, U/mL), 100 μM/mL ATP and 100 μCi/mL γ-³²P-ATP (Amersham, 9.25mBq/250 μCi/25 μL, activity >5000 Ci/mmol) in kinase buffer (50 mMtris-HCl, 150 mM NaCl, 30 mM MgCl₂, 4 mM DTT, 2 mM EGTA, pH 7.5) wasapplied by capillary forces into the gap formed by the second glasssurface lying on the modified glass surface. Incubation was then carriedout for 30 min at room temperature in an almost water-saturatedatmosphere. In order to reduce the background caused by the non-specificbinding of ATP or kinase molecules to the glass surfaces, the modifiedglass surface was washed as follows:

[0233] three times for 3 minutes at room temperature using washingbuffer (1% SDS and 1% Tween20 in 50 mM TRIS buffer, pH 7.5, 200 mM NaCl)

[0234] twice for 3 minutes at room temperature using 1M NaCl solution

[0235] twice for 3 minutes at room temperature using distilled water

[0236] twice for 3 minutes at room temperature using 80% formic acid inethanol

[0237] twice for 3 minutes at room temperature using distilled water

[0238] twice for 5 minutes at 50° C. using a solution containing 6Murea, 2M thiourea and 1% SDS

[0239] three times for 3 minutes at room temperature using distilledwater

[0240] three times for 3 minutes at room temperature using methanol

[0241] After drying the glass surface the quantity of radioactivephosphate incorporated in the glass-surface-bound peptides wasdetermined using a PhosphorImager system (FLA-3000, FUJIFILM) (see FIG.7).

Example 28 Analysis of Kinase-Mediated Peptide Modifications on ModifiedGlass Surfaces (see FIG. 6)

[0242] A glass surface modified with the peptideLeu-Arg-Arg-Ala-Ser-Leu-Gly-thioamide (see Example 24) was incubatedwith 10 mL of 100 μM ATP in kinase buffer (50 mM tris-HCl, 150 mM NaCl,30 mM MgCl₂, 4 mM DTT, 2 mM EGTA, pH 7.5) for 10 minutes at roomtemperature. The glass surface was dried and a second unmodified glasssurface of the same dimensions was then placed on the peptide-modifiedglass surface. Then 50 μL of a mixture of protein kinase A (Sigma,P26452, U/mL), 100 μM/mL ATP and 100 μCi/mL γ-³²P-ATP (Amersham, 9.25mBq/250 μCi/25 μL, activity >5000 Ci/mmol) in kinase buffer (50 mMtris-HCl, 150 mM NaCl, 30 mM MgCl₂, 4 mM DTT, 2 mM EGTA, pH 7.5) wasapplied by capillary forces into the gap formed by the second glasssurface lying on the modified glass surface. Incubation was then carriedout for 30 min at room temperature in an almost water-saturatedatmosphere. In order to reduce the background caused by the non-specificbinding of ATP or kinase molecules to the glass surfaces, the modifiedglass surface was washed as follows:

[0243] three times for 3 minutes at room temperature using washingbuffer (1% SDS and 1% Tween20 in 50 mM TRIS buffer, pH 7.5, 200 mM NaCl)

[0244] twice for 3 minutes at room temperature using 1M NaCl solution

[0245] twice for 3 minutes at room temperature using distilled water

[0246] twice for 3 minutes at room temperature using 80% formic acid inethanol

[0247] twice for 3 minutes at room temperature using distilled water

[0248] twice for 5 minutes at 50° C. using a solution containing 6Murea, 2M thiourea and 1% SDS

[0249] three times for 3 minutes at room temperature using distilledwater three times for 3 minutes at room temperature using methanol

[0250] After drying the glass surface the quantity of radioactivephosphate incorporated in the glass-surface-bound peptides wasdetermined using a PhosphorImager system (FLA-3000, FUJIFILM) (see FIG.6).

Example 29 Analysis of Kinase-Mediated Peptide Modifications on ModifiedGlass Surfaces (see FIG. 1)

[0251] A glass surface (maleinimido-functionalised glass surface, seeExample 2) modified with the peptidesCys-βAla-Leu-Arg-Arg-Ala-Ser-Leu-Gly-NH₂,Cys-βAla-Gln-Lys-Arg-Pro-Ser-Gln-Arg-Ser-Lys-NH₂ andCys-βAla-Arg-Arg-Lys-Asp-Leu-His-Ap-Arg-Glu-Glu-Asp-Glu-Ala-Met-Ser-Ile-Thr-Ala-NH₂or the corresponding control peptidesCys-βAla-Leu-Arg-Arg-Ala-Ala-Leu-Gly-NH₂,Cys-βAla-Gln-Lys-Arg-Pro-Ala-Gln-Arg-Ala-Lys-NH₂ andCys-βAla-Arg-Arg-Lys-Asp-Leu-His-Asp-Asp-Glu-Glu-Asp-Glu-Ala-Met-Ala-Ile-Ala-Ala-NH₂(see Example 22) was incubated with 10 mL of 100 μM ATP in kinase buffer(50 mM tris-HCl, 150 mM NaCl, 30 mM MgCl₂, 4 mM DTT, 2 mM EGTA, pH 7.5)for 10 minutes at room temperature. Then, 1 μL of a mixture of proteinkinase A (Protein kinase A, Sigma, P2645, 1.67 μg/mL forCys-βAla-Leu-Arg-Arg-Ala-Ser-Leu-Gly-NH₂ andCys-βAla-Leu-Arg-Arg-Ala-Ala-Leu-Gly-NH₂, Protein kinase C, Sigma,P7956, 1.3 μg/mL for Cys-βAla-Gln-Lys-Arg-Pro-Ser-Gln-Arg-Ser-Lys-NH₂and Cys-βAla-Gln-Lys-Arg-Pro-Ala-Gln-Arg-Ala-Lys-NH₂; caseinkinase 1,New England Biolabs, P6030S, 2.5 μg/mL forCys-βAla-Arg-Arg-Lys-Asp-Leu-His-Asp-Asp-Glu-Glu-Asp-Glu-Ala-Met-Ser-Ile-Thr-Ala-NH₂andCys-βAla-Arg-Arg-Lys-Asp-Leu-His-Asp-Asp-Glu-Glu-Asp-Glu-Ala-Met-Ala-Ile-Ala-Ala-NH₂),100 μM/mL ATP and 100 μCi/mL γ-³²P-ATP (Amersham, 9.25 mBq/250 μCi/25μL, activity >5000 Ci/mmol) in kinase buffer (50 mM tris-HCl, 150 mMNaCl, 30 mM MgCl₂, 4 mM DTT, 2 mM EGTA, pH 7.5) was spotted onto thepeptide-modified glass surfaces and incubated for 30 min at roomtemperature in an almost water-saturated atmosphere. In order to reducethe background caused by the non-specific binding of ATP or kinasemolecules to the glass surfaces, the modified glass surfaces were washedas follows:

[0252] three times for 3 minutes at room temperature using washingbuffer (1% SDS and 1% Tween20 in 50 mM TRIS buffer, pH 7.5, 200 mM NaCl)

[0253] twice for 3 minutes at room temperature using 1M NaCl solution

[0254] twice for 3 minutes at room temperature using distilled water

[0255] twice for 3 minutes at room temperature using 80% formic acid inethanol

[0256] twice for 3 minutes at room temperature using distilled water

[0257] twice for 5 minutes at 50° C. using a solution containing 6Murea, 2M thiourea and 1% SDS

[0258] three times for 3 minutes at room temperature using distilledwater

[0259] three times for 3 minutes at room temperature using methanol

[0260] After drying the glass surface the quantity of radioactivephosphate incorporated in the glass-surface-bound peptides wasdetermined using a PhosphorImager system (FLA-3000, FUJIFILM) (see FIG.1).

Example 30 Analysis of Kinase-Mediated Peptide Modifications on ModifiedGlass Surfaces (see FIG. 2)

[0261] Surfaces (maleinimido-functionalised glass surface, see Example1; maleinimidobutyryl-β-alaninine-functionalised cellulose andmaleinimidobutyryl-β-alaninine-functionalised polypropylene membranes)modified with the peptide Cys-βAla-Leu-Arg-Arg-Ala-Ser-Leu-Gly-NH₂ andCys-βAla-Leu-Arg-Arg-Ala-Ala-Leu-Gly-NH₂ were incubated with 10 mL of100 μM ATP in kinase buffer (50 mM tris-HCl, 150 mM NaCl, 30 mM MgCl₂, 4mM DTT, 2 mM EGTA, pH 7.5) for 10 minutes at room temperature. Thesurfaces were then coated with a mixture of protein kinase A (Sigma,P26452, U/mL), 100 μM/mL ATP and 100 μCi/mL γ-³²P-ATP (Amersham, 9.25mBq/250 μCi/25 μL, activity >5000 Ci/mmol) in kinase buffer (50 mMtris-HCl, 150 mM NaCl, 30 mM MgCl₂, 4 mM DTT, 2 mM EGTA, pH 7.5) andincubated for 30 min at room temperature in an almost water-saturatedatmosphere. In order to reduce the background caused by the non-specificbinding of ATP or kinase molecules to the glass surfaces, the modifiedglass surfaces were washed as follows:

[0262] three times for 3 minutes at room temperature using washingbuffer (1% SDS and 1% Tween20 in 50 mM TRIS buffer, pH 7.5, 200 mM NaCl)

[0263] twice for 3 minutes at room temperature using 1M NaCl solution

[0264] twice for 3 minutes at room temperature using distilled water

[0265] twice for 3 minutes at room temperature using 80% formic acid inethanol

[0266] twice for 3 minutes at room temperature using distilled water

[0267] twice for 5 minutes at 50° C. using a solution containing 6Murea, 2M thiourea and 1% SDS

[0268] three times for 3 minutes at room temperature using distilledwater

[0269] three times for 3 minutes at room temperature using methanol

[0270] After drying the glass surface the quantity of radioactivephosphate incorporated in the glass-surface-bound peptides wasdetermined using a PhosphorImager system (FLA-3000, FUJIFILM) (see FIG.2).

Example 31 Analysis of Kinase-Mediated Peptide Modifications on VariousModified Glass Surfaces (see FIG. 3)

[0271] Surfaces (maleinimido-functionalised glass surface, see Example 1and maleinimidobutyryl-α-alaninine-functionalised cellulose) modifiedwith the peptide Cys-Ala-Leu-Arg-Arg-Ala-Ser-Leu-Gly-NH₂ were incubatedwith 10 mL of 100 μM ATP in kinase buffer (50 mM tris-HCl, 150 mM NaCl,30 mM MgCl₂, 4 mM DTT, 2 mM EGTA, pH 7.5) for 10 minutes at roomtemperature. The surfaces were then mixed with a mixture of proteinkinase A (Sigma, P26452, U/mL), 100 μM/mL ATP and 100 μCi/mL γ-³²P-ATP(Amersham, 9.25 mBq/250 μCi/25 μL, activity >5000 Ci/mmol) in kinasebuffer (50 mM tris-HCl, 150 mM NaCl, 30 mM MgCl₂, 4 mM DTT, 2 mM EGTA,pH 7.5) and incubated for the specified times at room temperature in analmost water-saturated atmosphere. In order to reduce the backgroundcaused by the non-specific binding of ATP or kinase molecules to theglass surfaces, the modified glass surfaces were washed as follows:

[0272] three times for 3 minutes at room temperature using washingbuffer (1% SDS and 1% Tween20 in 50 mM TRIS buffer, pH 7.5, 200 mM NaCl)

[0273] twice for 3 minutes at room temperature using 1M NaCl solution

[0274] twice for 3 minutes at room temperature using distilled water

[0275] twice for 3 minutes at room temperature using 80% formic acid inethanol

[0276] twice for 3 minutes at room temperature using distilled water

[0277] twice for 5 minutes at 50° C. using a solution containing 6Murea, 2M thiourea and 1% SDS

[0278] three times for 3 minutes at room temperature using distilledwater three times for 3 minutes at room temperature using methanol

[0279] After drying the glass surface the quantity of radioactivephosphate incorporated in the glass-surface-bound peptides wasdetermined using a PhosphorImager system (FLA-3000, FUJIFILM) (see FIG.3).

Example 32 Analysis of Kinase-Mediated Peptide Modifications on VariousModified Glass Surfaces (see FIG. 8)

[0280] Precisely 43 serine- and/or threonine-containing peptides(potential substrate peptides for kinases) and the corresponding controlpeptides, each modified at the N-terminus with the dipeptidecysteinyl-β-alanine, were coupled to a maleinimido-functionalised glasssurface by a Michael addition (see Example 18). The assembly of thepeptides is shown in FIG. 8A. The numbering of the spots can be seenfrom FIG. 8C and the sequences of the peptides used are obtained fromExample 18. After application of the peptide, the modified glass surfacewas first pre-incubated using 10 mL of 100 μM ATP ATP solution in 50 mMsodium phosphate buffer pH 7.5 for 10 for 10 minutes. The modified glasssurface was then covered with a cover glass and protein kinase C (10U/mL) together with ATP/γ-³²P-ATP mixture (100 μM/mL; 100 μCi/mL) wasincorporated in the intermediate space formed by means of capillaryforce. Incubation was then carried out for 30 min at 25° C. In order toreduce the background caused by the non-specific binding of ATP orkinase molecules to the glass surfaces, the modified glass surfaces werewashed as follows:

[0281] three times for 3 minutes at room temperature using washingbuffer (1% SDS and 1% Tween20 in 50 mM TRIS buffer, pH 7.5, 200 mM NaCl)

[0282] twice for 3 minutes at room temperature using 1M NaCl solution

[0283] twice for 3 minutes at room temperature using distilled water

[0284] twice for 3 minutes at room temperature using 80% formic acid inethanol

[0285] twice for 3 minutes at room temperature using distilled water

[0286] twice for 5 minutes at 50° C. using a solution containing 6Murea, 2M thiourea and 1% SDS

[0287] three times for 3 minutes at room temperature using distilledwater

[0288] three times for 3 minutes at room temperature using methanol

[0289] After drying the glass surface the quantity of radioactivephosphate incorporated in the glass-surface-bound peptides wasdetermined using a PhosphorImager system (FLA-3000, FUJIFILM). Theresulting image is shown in FIG. 8B. The spots of higher signalintensity in all three subarrays were assigned to the correspondingpeptides phosphorylated by the protein kinase C. Their primarystructures are shown in FIG. 8D. It is clear that the peptides known asprotein kinase C substrates (substrate peptides Nos. 3, 23, 27, 41, 43)and other peptides not described as substrates for protein kinase C arerecognised and phosphorylated by this kinase on the modified glasssurface. It is thus clear that such an assembly is suitable forcharacterising the substrate specificity of a kinase, such as proteinkinase C for example.

Example 33 Analysis of Kinase-Mediated Peptide Modifications on VariousModified Glass Surfaces (see FIG. 9)

[0290] Precisely 43 serine- and/or threonine-containing peptides(potential substrate peptides for kinases) and the corresponding controlpeptides, each modified at the N-terminus with the dipeptidecysteinyl-β-alanine, were coupled to a maleinimido-functionalised glasssurface by a Michael addition (see Example 18). The assembly of thepeptides is shown in FIG. 8A. The numbering of the spots can be seenfrom FIG. 8C and the sequences of the peptides used are obtained fromExample 18. After application of the peptide, the modified glass surfacewas first pre-incubated using 10 mL of 100 μM ATP solution in 50 mMsodium phosphate buffer pH 7.5 for 10 for 10 minutes. The modified glasssurface was then covered with a cover glass and protein kinase A (10U/mL) together with ATP/γ-³²P-ATP mixture (100 μM/mL; 100 μCi/mL) wasincorporated in the intermediate space formed by means of capillaryforce. Incubation was then carried out for 30 min at 25° C. In order toreduce the background caused by the non-specific binding of ATP orkinase molecules to the glass surfaces, the modified glass surfaces werewashed as follows:

[0291] three times for 3 minutes at room temperature using washingbuffer (1% SDS and 1% Tween20 in 50 mM TRIS buffer, pH 7.5, 200 mM NaCl)

[0292] twice for 3 minutes at room temperature using 1M NaCl solution

[0293] twice for 3 minutes at room temperature using distilled water

[0294] twice for 3 minutes at room temperature using 80% formic acid inethanol

[0295] twice for 3 minutes at room temperature using distilled water

[0296] twice for 5 minutes at 50° C. using a solution containing 6Murea, 2M thiourea and 1% SDS

[0297] three times for 3 minutes at room temperature using distilledwater three times for 3 minutes at room temperature using methanol

[0298] After drying the glass surface the quantity of radioactivephosphate incorporated in the glass-surface-bound peptides wasdetermined using a PhosphorImager system (FLA-3000, FUJIFILM). Theresulting image is shown in FIG. 9B. The spots of higher signalintensity in all three subarrays were assigned to the correspondingpeptides phosphorylated by the protein kinase A. Their primarystructures are shown in FIG. 9D. It is clear that with one exception,all the peptides on the modified glass surface are recognised andphosphorylated by protein kinase A which carries two arginine residuesin position −2 and −3 (N-terminal) to the serine. This sequence motifRRxS is described as a preferred substrate motif for protein kinase A(A. Kreegipuu, N. Blom, S. Brunak, J. Jarv, 1998, Statistical analysisof protein kinase specificity determinants, FEBS Lett., 430, 45-50). Thepeptide 83 is probably not phosphorylated because of the excessiveN-terminal localisation of the substrate motif. It is thus clear thatsuch an assembly is suitable for characterising the substratespecificity of a kinase, such as protein kinase A for example.

Example 34 Analysis of Kinase-Mediated Peptide Modifications on VariousModified Glass Surfaces (see FIG. 10)

[0299] Precisely 79 peptides, each modified at the N-terminus with thedipeptide amino-oxyacetic acid-β-alanine, were coupled to analdehyde-functionalised glass surface (see Example 20). The primarystructure of the MBP is shown in FIG. 10C. For the residues shown inbold print a phosphorylation by protein kinase A was described in theprior art (A. Kishimoto, K. Nishiyama, H. Nakanishi, Y. Uratsuji, H.Numura, Y. Takeyama, Y. Nishizuka, 1985, Studies on the phosphorylationof myelin basic protein by protein kinase C and adenosine3′:5′-monophosphate-dependent protein kinase, J. Biol. Chem., 260,12492-12499). The 13-mer peptides in the scan show a sequence shift oftwo amino acids. The peptide assembly is shown in FIG. 10B. Thus,peptide No. 1 represents the amino acid sequence 1-13 of the primarystructure of MBP, peptide No. 2 represents the amino acid sequence 3-15of the primary structure of MBP, etc. Three identical subarrays areapplied to the glass surface. One of these subarrays is shown in FIG.10A. After application of the peptides, the modified glass surface wasfirst pre-incubated for 10 minutes using 10 ml of 100 μM ATP solution in50 mM of sodium phosphate buffer pH 7.5. The modified glass surface wasthen covered with a cover glass and Protein kinase A (10 U/mL) togetherwith ATP/γ³²P-ATP mixture (100 μM/mL; 100 μCi/mL) was then inserted intothe intermediate space formed thereby by means of capillary force.Incubation was then carried out for 30 min at 25° C. In order to reducethe background caused by the non-specific binding of ATP or kinasemolecules to the glass surfaces, the modified glass surfaces were washedas follows:

[0300] three times for 3minutes at room temperature using washing buffer(1% SDS and 1% Tween20 in 50 mM TRIS buffer, pH 7.5, 200 mM NaCl)

[0301] twice for 3 minutes at room temperature using 1M NaCl solution

[0302] twice for 3 minutes at room temperature using distilled water

[0303] twice for 3 minutes at room temperature using 80% formic acid inethanol

[0304] twice for 3 minutes at room temperature using distilled water

[0305] twice for 5 minutes at 50° C. using a solution containing 6Murea, 2M thiourea and 1% SDS

[0306] three times for 3 minutes at room temperature using distilledwater

[0307] three times for 3 minutes at room temperature using methanol

[0308] After drying the glass surface the quantity of radioactivephosphate incorporated in the glass-surface-bound peptides wasdetermined using a PhosphorImager system (FLA-3000, FUJIFILM). Theresulting image is shown in FIG. 10A. The spots of higher signalintensity in all three subarrays were assigned to the correspondingpeptides phosphorylated by the protein kinase A. Their primarystructures are shown in FIG. 10D. It is clear that most of the peptideson the modified glass surface are recognised and phosphorylated byprotein kinase A which was also found in the experiment carried out insolution (A. Kishimoto, K. Nishiyama, H. Nakanishi, Y. Uratsuji, H.Numura, Y. Takeyama, Y. Nishizuka, 1985, Studies on the phosphorylationof myelin basic protein by protein kinase C and adenosine3′:5′-monophosphate-dependent protein kinase, J. Biol. Chem., 260,12492-12499).

[0309] The features of the invention disclosed in the previousdescription, the examples, the claims, the drawings and the sequenceprotocol can both individually and in any combination be important forthe implementation of the invention in its various embodiments.

1 144 1 4 PRT Unknown cell-adhesive peptide 1 Arg Gly Asp Cys 1 2 18 PRTUnknown kinase substrate 2 Arg Arg Lys Asp Leu His Asp Asp Glu Glu AspGlu Ala Met Ser Ile 1 5 10 15 Thr Ala 3 9 PRT Unknown kinase substrate 3Gln Lys Arg Pro Ser Gln Arg Ser Lys 1 5 4 7 PRT Unknown kinase substrate4 Leu Arg Arg Ala Ser Leu Gly 1 5 5 7 PRT Unknown peptide/ controlpeptide 5 Leu Arg Arg Ala Ser Leu Gly 1 5 6 7 PRT Unknown peptide/control peptide 6 Leu Arg Arg Ala Ala Leu Gly 1 5 7 7 PRT Unknownpeptide/ control peptide 7 Leu Arg Arg Ala Ser Leu Gly 1 5 8 8 PRTUnknown peptide/ control peptide 8 Xaa Leu Arg Arg Ala Ser Leu Gly 1 5 911 PRT Unknown protein kinase C substrate 9 Ala Lys Arg Arg Arg Leu SerSer Leu Arg Ala 1 5 10 10 12 PRT Unknown protein kinase C substrate 10Pro Leu Ser Arg Thr Leu Ser Val Ala Ala Lys Lys 1 5 10 11 11 PRT Unknownprotein kinase C subtstrate 11 Gln Lys Arg Pro Ser Gln Arg Ser Lys TyrLeu 1 5 10 12 12 PRT Unknown protein kinase C substrate 12 Lys Lys ArgPhe Ser Phe Lys Lys Ser Phe Lys Leu 1 5 10 13 9 PRT Unknown proteinkinase C substrate 13 Pro Lys Asp Pro Ser Gln Arg Arg Arg 1 5 14 9 PRTUnknown not protein kinase C substrate 14 Gly Arg Thr Gly Arg Arg AsnSer Ile 1 5 15 10 PRT Unknown not protein kinase C substrate 15 Asp AspAsp Glu Glu Ser Ile Thr Arg Arg 1 5 10 16 9 PRT Unknown not proteinkinase C substrate 16 Glu Arg Ser Pro Ser Pro Ser Phe Arg 1 5 17 11 PRTUnknown not protein kinase C substrate 17 Gly Arg Pro Arg Thr Ser SerPhe Ala Glu Gly 1 5 10 18 13 PRT Unknown not protein kinase C substrate18 Lys Lys Lys Ala Leu Ser Arg Gln Leu Ser Val Ala Ala 1 5 10 19 10 PRTUnknown not protein kinase C substrate 19 Lys Lys Leu Asn Arg Thr LeuSer Val Ala 1 5 10 20 13 PRT Unknown not protein kinase C substrate 20Lys Arg Arg Glu Ile Leu Ser Arg Arg Pro Ser Tyr Arg 1 5 10 21 10 PRTUnknown not protein kinase C substrate 21 Pro Leu Ser Arg Thr Leu SerVal Ser Ser 1 5 10 22 7 PRT Unknown not protein kinase C substrate 22Arg Pro Arg Ala Ala Thr Phe 1 5 23 13 PRT Unknown not protein kinase Csubstrate 23 Arg Phe Ala Arg Lys Gly Ser Leu Arg Gln Lys Asn Val 1 5 1024 9 PRT Unknown not protein kinase C substrate 24 Arg Glu Ala Arg SerArg Ala Ser Thr 1 5 25 13 PRT Unknown substrate peptide 25 Cys Xaa AlaLys Arg Arg Arg Leu Ser Ser Leu Arg Ala 1 5 10 26 11 PRT Unknownsubstrate peptide 26 Cys Xaa Gly Arg Thr Gly Arg Arg Asn Ser Ile 1 5 1027 13 PRT Unknown substrate peptide 27 Cys Xaa Gly Arg Pro Arg Thr SerSer Phe Ala Glu Gly 1 5 10 28 15 PRT Unknown substrate peptide 28 CysXaa Lys Arg Arg Glu Ile Leu Ser Arg Arg Pro Ser Tyr Arg 1 5 10 15 29 13PRT Unknown substrate peptide 29 Cys Xaa Gln Lys Arg Pro Ser Gln Arg SerLys Tyr Leu 1 5 10 30 15 PRT Unknown substrate peptide 30 Cys Xaa ArgPhe Ala Arg Lys Gly Ser Leu Arg Gln Lys Asn Val 1 5 10 15 31 9 PRTUnknown substrate peptide 31 Cys Xaa Leu Arg Arg Ala Ser Leu Gly 1 5 32169 PRT Bos taurus 32 Ala Ala Gln Lys Arg Pro Ser Gln Arg Ser Lys TyrLeu Ala Ser Ala 1 5 10 15 Ser Thr Met Asp His Ala Arg His Gly Phe LeuPro Arg His Arg Asp 20 25 30 Thr Gly Ile Leu Asp Ser Leu Gly Arg Phe PheGly Ser Asp Arg Gly 35 40 45 Ala Pro Lys Arg Gly Ser Gly Lys Asp Gly HisHis Ala Ala Arg Thr 50 55 60 Thr His Tyr Gly Ser Leu Pro Gln Lys Ala GlnGly His Arg Pro Gln 65 70 75 80 Asp Glu Asn Pro Val Val His Phe Phe LysAsn Ile Val Thr Pro Arg 85 90 95 Thr Pro Pro Pro Ser Gln Gly Lys Gly ArgGly Leu Ser Leu Ser Arg 100 105 110 Phe Ser Trp Gly Ala Glu Gly Gln LysPro Gly Phe Gly Tyr Gly Gly 115 120 125 Arg Ala Ser Asp Tyr Lys Ser AlaHis Lys Gly Leu Lys Gly His Asp 130 135 140 Ala Gln Gly Thr Leu Ser LysIle Phe Lys Leu Gly Gly Arg Asp Ser 145 150 155 160 Arg Ser Gly Ser ProMet Ala Arg Arg 165 33 15 PRT Unknown substrate peptide 33 Xaa Xaa AlaAla Gln Lys Arg Pro Ser Gln Arg Ser Lys Tyr Leu 1 5 10 15 34 15 PRTUnknown substrate peptide 34 Xaa Xaa Gln Lys Arg Pro Ser Gln Arg Ser LysTyr Leu Ala Ser 1 5 10 15 35 15 PRT Unknown substrate peptide 35 Xaa XaaArg Pro Ser Gln Arg Ser Lys Tyr Leu Ala Ser Ala Ser 1 5 10 15 36 15 PRTUnknown substrate peptide 36 Xaa Xaa Phe Gly Ser Asp Arg Gly Ala Pro LysArg Gly Ser Gly 1 5 10 15 37 15 PRT Unknown substrate peptide 37 Xaa XaaSer Asp Arg Gly Ala Pro Lys Arg Gly Ser Gly Lys Asp 1 5 10 15 38 15 PRTUnknown substrate peptide 38 Xaa Xaa Arg Gly Ala Pro Lys Arg Gly Ser GlyLys Asp Gly His 1 5 10 15 39 15 PRT Unknown substrate peptide 39 Xaa XaaAla Pro Lys Arg Gly Ser Gly Lys Asp Gly His His Ala 1 5 10 15 40 15 PRTUnknown substrate peptide 40 Xaa Xaa Lys Arg Gly Ser Gly Lys Asp Gly HisHis Ala Ala Arg 1 5 10 15 41 15 PRT Unknown substrate peptide 41 Xaa XaaGly Ser Gly Lys Asp Gly His His Ala Ala Arg Thr Thr 1 5 10 15 42 15 PRTUnknown substrate peptide 42 Xaa Xaa Pro Pro Ser Gln Gly Lys Gly Arg GlyLeu Ser Leu Ser 1 5 10 15 43 15 PRT Unknown substrate peptide 43 Xaa XaaSer Gln Gly Lys Gly Arg Gly Leu Ser Leu Ser Arg Phe 1 5 10 15 44 15 PRTUnknown substrate peptide 44 Xaa Xaa Gly Lys Gly Arg Gly Leu Ser Leu SerArg Phe Ser Trp 1 5 10 15 45 15 PRT Unknown substrate peptide 45 Xaa XaaGly Gly Arg Ala Ser Asp Tyr Lys Ser Ala His Lys Gly 1 5 10 15 46 15 PRTUnknown substrate peptide 46 Xaa Xaa Arg Ala Ser Asp Tyr Lys Ser Ala HisLys Gly Leu Lys 1 5 10 15 47 15 PRT Unknown synthesized peptide sequence47 Cys Xaa Lys Lys Ala Leu Arg Arg Gln Glu Thr Val Asp Ala Leu 1 5 10 1548 15 PRT Unknown synthesized peptide sequence 48 Cys Xaa Lys Lys AlaLeu Arg Arg Gln Glu Ala Val Asp Ala Leu 1 5 10 15 49 13 PRT Unknownsynthesized peptide sequence 49 Cys Xaa Ala Lys Arg Arg Arg Leu Ser SerLeu Arg Ala 1 5 10 50 13 PRT Unknown synthesized peptide sequence 50 CysXaa Ala Lys Arg Arg Arg Leu Ala Ala Leu Arg Ala 1 5 10 51 11 PRT Unknownsynthesized peptide sequence 51 Cys Xaa Gly Arg Thr Gly Arg Arg Asn SerIle 1 5 10 52 11 PRT Unknown synthesized peptide sequence 52 Cys Xaa GlyArg Ala Gly Arg Arg Asn Ala Ile 1 5 10 53 12 PRT Unknown synthesizedpeptide sequence 53 Cys Xaa Asp Asp Asp Glu Glu Ser Ile Thr Arg Arg 1 510 54 12 PRT Unknown synthesized peptide sequence 54 Cys Xaa Asp Asp AspGlu Glu Ala Ile Ala Arg Arg 1 5 10 55 11 PRT Unknown synthesized peptidesequence 55 Cys Xaa Glu Arg Ser Pro Ser Pro Ser Phe Arg 1 5 10 56 11 PRTUnknown synthesized peptide sequence 56 Cys Xaa Glu Arg Ala Pro Ala ProAla Phe Arg 1 5 10 57 13 PRT Unknown synthesized peptide sequence 57 CysXaa Gly Arg Pro Arg Thr Ser Ser Phe Ala Glu Gly 1 5 10 58 13 PRT Unknownsynthesized peptide sequence 58 Cys Xaa Gly Arg Pro Arg Ala Ala Ala PheAla Glu Gly 1 5 10 59 15 PRT Unknown synthesized peptide sequence 59 CysXaa Lys Lys Lys Ala Leu Ser Arg Gln Leu Ser Val Ala Ala 1 5 10 15 60 15PRT Unknown synthesized peptide sequence 60 Cys Xaa Lys Lys Lys Ala LeuAla Arg Gln Leu Ala Val Ala Ala 1 5 10 15 61 12 PRT Unknown synthesizedpeptide sequence 61 Cys Xaa Lys Lys Leu Asn Arg Thr Leu Ser Val Ala 1 510 62 12 PRT Unknown synthesized peptide sequence 62 Cys Xaa Lys Lys LeuAsn Arg Ala Leu Ala Val Ala 1 5 10 63 11 PRT Unknown synthesized peptidesequence 63 Cys Xaa Lys Arg Gln Gln Ser Phe Asp Leu Phe 1 5 10 64 11 PRTUnknown synthesized peptide sequence 64 Cys Xaa Lys Arg Gln Gln Ala PheAsp Leu Phe 1 5 10 65 15 PRT Unknown synthesized peptide sequence 65 CysXaa Lys Arg Arg Glu Ile Leu Ser Arg Arg Pro Ser Tyr Arg 1 5 10 15 66 15PRT Unknown synthesized peptide sequence 66 Cys Xaa Lys Arg Arg Glu IleLeu Ala Arg Arg Pro Ala Phe Arg 1 5 10 15 67 11 PRT Unknown synthesizedpeptide sequence 67 Cys Xaa Leu Arg Ala Pro Ser Trp Ile Asp Thr 1 5 1068 11 PRT Unknown synthesized peptide sequence 68 Cys Xaa Leu Arg AlaPro Ala Trp Ile Asp Ala 1 5 10 69 14 PRT Unknown synthesized peptidesequence 69 Cys Xaa Pro Leu Ser Arg Thr Leu Ser Val Ala Ala Lys Lys 1 510 70 14 PRT Unknown synthesized peptide sequence 70 Cys Xaa Pro Leu AlaArg Ala Leu Ala Val Ala Ala Lys Lys 1 5 10 71 12 PRT Unknown synthesizedpeptide sequence 71 Cys Xaa Pro Leu Ser Arg Thr Leu Ser Val Ser Ser 1 510 72 12 PRT Unknown synthesized peptide sequence 72 Cys Xaa Pro Leu AlaArg Ala Leu Ala Val Ala Ala 1 5 10 73 13 PRT Unknown synthesized peptidesequence 73 Cys Xaa Gln Lys Arg Pro Ser Gln Arg Ser Lys Tyr Leu 1 5 1074 13 PRT Unknown synthesized peptide sequence 74 Cys Xaa Gln Lys ArgPro Ala Gln Arg Ala Lys Phe Leu 1 5 10 75 10 PRT Unknown synthesizedpeptide sequence 75 Cys Xaa Arg Lys Ile Ser Ala Ser Glu Phe 1 5 10 76 10PRT Unknown synthesized peptide sequence 76 Cys Xaa Arg Lys Ile Ala AlaAla Glu Phe 1 5 10 77 12 PRT Unknown synthesized peptide sequence 77 ProLys Thr Pro Lys Lys Ala Lys Lys Leu Xaa Cys 1 5 10 78 12 PRT Unknownsynthesized peptide sequence 78 Pro Lys Ala Pro Lys Lys Ala Lys Lys LeuXaa Cys 1 5 10 79 9 PRT Unknown synthesized peptide sequence 79 Cys XaaArg Pro Arg Ala Ala Thr Phe 1 5 80 9 PRT Unknown synthesized peptidesequence 80 Cys Xaa Arg Pro Arg Ala Ala Ala Phe 1 5 81 10 PRT Unknownsynthesized peptide sequence 81 Cys Xaa Arg Arg Arg Ala Pro Leu Ser Pro1 5 10 82 10 PRT Unknown synthesized peptide sequence 82 Cys Xaa Arg ArgArg Ala Pro Leu Ala Pro 1 5 10 83 12 PRT Unknown synthesized peptidesequence 83 Cys Xaa Arg Arg Arg Glu Glu Glu Thr Glu Glu Glu 1 5 10 84 12PRT Unknown synthesized peptide sequence 84 Cys Xaa Arg Arg Arg Glu GluGlu Ala Glu Glu Glu 1 5 10 85 13 PRT Unknown synthesized peptidesequence 85 Cys Xaa Met His Arg Gln Glu Thr Val Asp Cys Leu Lys 1 5 1086 13 PRT Unknown synthesized peptide sequence 86 Cys Xaa Met His ArgGln Glu Ala Val Asp Cys Leu Lys 1 5 10 87 14 PRT Unknown synthesizedpeptide sequence 87 Cys Xaa Lys Lys Arg Phe Ser Phe Lys Lys Ser Phe LysLeu 1 5 10 88 14 PRT Unknown synthesized peptide sequence 88 Cys Xaa LysLys Arg Phe Ala Phe Lys Lys Ala Phe Lys Leu 1 5 10 89 11 PRT Unknownsynthesized peptide sequence 89 Cys Xaa Pro Lys Asp Pro Ser Gln Arg ArgArg 1 5 10 90 11 PRT Unknown synthesized peptide sequence 90 Cys Xaa ProLys Asp Pro Ala Gln Arg Arg Arg 1 5 10 91 11 PRT Unknown synthesizedpeptide sequence 91 Cys Xaa Ile Ala Ala Asp Ser Glu Ala Glu Gln 1 5 1092 11 PRT Unknown synthesized peptide sequence 92 Cys Xaa Ile Ala AlaAsp Ala Glu Ala Glu Gln 1 5 10 93 11 PRT Unknown synthesized peptidesequence 93 Cys Xaa Ser Pro Ala Leu Thr Gly Asp Glu Ala 1 5 10 94 11 PRTUnknown synthesized peptide sequence 94 Cys Xaa Ala Pro Ala Leu Ala GlyAsp Glu Ala 1 5 10 95 11 PRT Unknown synthesized peptide sequence 95 CysXaa Gly Arg Ile Leu Thr Leu Pro Arg Ser 1 5 10 96 11 PRT Unknownsynthesized peptide sequence 96 Cys Xaa Gly Arg Ile Leu Ala Leu Pro ArgAla 1 5 10 97 11 PRT Unknown synthesized peptide sequence 97 Cys Xaa MetGly Glu Ala Ser Gly Cys Gln Leu 1 5 10 98 11 PRT Unknown synthesizedpeptide sequence 98 Cys Xaa Met Gly Glu Ala Ala Gly Cys Gln Leu 1 5 1099 11 PRT Unknown synthesized peptide sequence 99 Cys Xaa Glu Glu ThrPro Tyr Ser Tyr Pro Thr 1 5 10 100 11 PRT Unknown synthesized peptidesequence 100 Cys Xaa Glu Glu Ala Pro Phe Ser Phe Pro Ala 1 5 10 101 11PRT Unknown synthesized peptide sequence 101 Cys Xaa Gly Asn His Thr TyrGln Glu Ile Ala 1 5 10 102 11 PRT Unknown synthesized peptide sequence102 Cys Xaa Gly Asn His Ala Phe Gln Glu Ile Ala 1 5 10 103 11 PRTUnknown synthesized peptide sequence 103 Leu Arg Ser Pro Ser Trp Glu ProPhe Xaa Cys 1 5 10 104 11 PRT Unknown synthesized peptide sequence 104Leu Arg Ala Pro Ala Trp Glu Pro Phe Xaa Cys 1 5 10 105 11 PRT Unknownsynthesized peptide sequence 105 Ser Ser Pro Val Tyr Gln Asp Ala Val XaaCys 1 5 10 106 11 PRT Unknown synthesized peptide sequence 106 Ala AlaPro Val Phe Gln Asp Ala Val Xaa Cys 1 5 10 107 11 PRT Unknownsynthesized peptide sequence 107 Cys Xaa Ser Arg Thr Leu Ser Val Ser SerLeu 1 5 10 108 11 PRT Unknown synthesized peptide sequence 108 Cys XaaAla Arg Ala Leu Ala Val Ala Ala Leu 1 5 10 109 11 PRT Unknownsynthesized peptide sequence 109 Leu Ser Val Ser Ser Leu Pro Gly Leu XaaCys 1 5 10 110 11 PRT Unknown synthesized peptide sequence 110 Leu SerVal Ala Ala Leu Pro Gly Leu Xaa Cys 1 5 10 111 11 PRT Unknownsynthesized peptide sequence 111 Cys Xaa Val Thr Pro Arg Thr Pro Pro ProSer 1 5 10 112 11 PRT Unknown synthesized peptide sequence 112 Cys XaaVal Ala Pro Arg Ala Pro Pro Pro Ala 1 5 10 113 15 PRT Unknownsynthesized peptide sequence 113 Cys Xaa Arg Phe Ala Arg Lys Gly Ser LeuArg Gln Lys Asn Val 1 5 10 15 114 15 PRT Unknown synthesized peptidesequence 114 Cys Xaa Arg Phe Ala Arg Lys Gly Ala Leu Arg Gln Lys Asn Val1 5 10 15 115 11 PRT Unknown synthesized peptide sequence 115 Cys XaaPro Arg Pro Ala Ser Val Pro Pro Ser 1 5 10 116 12 PRT Unknownsynthesized peptide sequence 116 Cys Xaa Pro Arg Pro Ala Ser Ala Val ProPro Ala 1 5 10 117 11 PRT Unknown synthesized peptide sequence 117 CysXaa Arg Glu Ala Arg Ser Arg Ala Ser Thr 1 5 10 118 11 PRT Unknownsynthesized peptide sequence 118 Cys Xaa Arg Glu Ala Arg Ala Arg Ala AlaAla 1 5 10 119 11 PRT Unknown synthesized peptide sequence 119 Gln SerTyr Ser Ser Ser Gln Arg Val Xaa Cys 1 5 10 120 11 PRT Unknownsynthesized peptide sequence 120 Gln Ser Tyr Ala Ala Ala Gln Arg Val XaaCys 1 5 10 121 11 PRT Unknown synthesized peptide sequence 121 Cys XaaGly Gly Gly Thr Ser Pro Val Phe Pro 1 5 10 122 11 PRT Unknownsynthesized peptide sequence 122 Cys Xaa Gly Gly Gly Ala Ala Pro Val PhePro 1 5 10 123 11 PRT Unknown synthesized peptide sequence 123 Leu TyrSer Ser Ser Pro Gly Gly Ala Xaa Cys 1 5 10 124 11 PRT Unknownsynthesized peptide sequence 124 Leu Tyr Ala Ala Ala Pro Gly Gly Ala XaaCys 1 5 10 125 11 PRT Unknown synthesized peptide sequence 125 Cys XaaAsp Leu Pro Leu Ser Pro Ser Ala Phe 1 5 10 126 11 PRT Unknownsynthesized peptide sequence 126 Cys Xaa Asp Leu Pro Leu Ala Pro Ala AlaPhe 1 5 10 127 11 PRT Unknown synthesized peptide sequence 127 Cys XaaThr Thr Pro Leu Ser Pro Thr Arg Leu 1 5 10 128 11 PRT Unknownsynthesized peptide sequence 128 Cys Xaa Ala Ala Pro Leu Ala Pro Ala ArgLeu 1 5 10 129 15 PRT Unknown synthesized peptide sequence 129 Arg ArgIle Ser Lys Asp Asn Pro Asp Tyr Gln Gln Asp Xaa Cys 1 5 10 15 130 15 PRTUnknown synthesized peptide sequence 130 Arg Arg Ile Ala Lys Asp Asn ProAsp Tyr Gln Gln Asp Xaa Cys 1 5 10 15 131 9 PRT Unknown synthesizedpeptide sequence 131 Cys Xaa Leu Arg Arg Ala Ser Leu Gly 1 5 132 9 PRTUnknown synthesized peptide sequence 132 Cys Xaa Leu Arg Arg Ala Ala LeuGly 1 5 133 9 PRT Unknown synthesized peptide sequence 133 Cys Xaa LeuArg Arg Ala Ser Leu Gly 1 5 134 11 PRT Unknown synthesized peptidesequence 134 Cys Xaa Gln Lys Arg Pro Ser Gln Arg Ser Lys 1 5 10 135 20PRT Unknown synthesized peptide sequence 135 Cys Xaa Arg Arg Lys Asp LeuHis Asp Arg Glu Glu Asp Glu Ala Met 1 5 10 15 Ser Ile Thr Ala 20 136 9PRT Unknown synthesized peptide sequence 136 Cys Xaa Leu Arg Arg Ala AlaLeu Gly 1 5 137 11 PRT Unknown synthesized peptide sequence 137 Cys XaaGln Lys Arg Pro Ala Gln Arg Ala Lys 1 5 10 138 20 PRT Unknownsynthesized peptide sequence 138 Cys Xaa Arg Arg Lys Asp Leu His Asp AspGlu Glu Asp Glu Ala Met 1 5 10 15 Ala Ile Ala Ala 20 139 9 PRT Unknownsynthesized peptide sequence 139 Cys Xaa Leu Arg Arg Ala Ser Leu Gly 1 5140 9 PRT Unknown synthesized peptide sequence 140 Cys Xaa Leu Arg ArgAla Ala Leu Gly 1 5 141 11 PRT Unknown synthesized peptide sequence 141Cys Xaa Gln Lys Arg Pro Ser Gln Arg Ser Lys 1 5 10 142 11 PRT Unknownsynthesized peptide sequence 142 Cys Xaa Gln Lys Arg Pro Ala Gln Arg AlaLys 1 5 10 143 20 PRT Unknown synthesized peptide sequence 143 Cys XaaArg Arg Lys Asp Leu His Asp Asp Glu Glu Asp Glu Ala Met 1 5 10 15 SerIle Thr Ala 20 144 20 PRT Unknown synthesized peptide sequence 144 CysXaa Arg Arg Lys Asp Leu His Asp Asp Glu Glu Asp Glu Ala Met 1 5 10 15Ala Ile Ala Ala 20

1. A method for determining the substrate specificity of an enzymaticactivity comprising the following steps: Providing an assemblycomprising a plurality of amino acid sequences on a planar surface of asupport material wherein the amino acid sequences are directionallyimmobilised, Contacting and/or incubating of an enzymatic activity withthe assembly, and Detecting a reaction between one or a plurality ofamino acid sequences immobilised on the assembly and the enzymaticactivity, characterised in that during the reaction of the enzymaticactivity with the assembly, a change in the molecular weight of at leastone of the amino acid sequences takes place.
 2. The method according toclaim 1, characterised in that the reaction is detected on or using theamino acid sequence immobilised on the surface of the support material.3. The method according to claim 1 or claim 2, characterised in that thechange in the molecular weight takes place by formation or cleaving of acovalent bond on one of the amino acid sequences, preferably on thatamino acid sequence which reacts with the enzymatic activity.
 4. Themethod according to any one of claims 1 to 3, characterised in that thereaction is detected by detecting the change in the molecular weight. 5.The method according to any one of claims 1 to 4, characterised in thatthe reaction is detected by a detection method selected from the groupcomprising autoradiography, plasmon resonance spectroscopy andfluorescence spectroscopy.
 6. The method according to any one of claims1 to 5, characterised in that at least one of the amino acid sequencesis a substrate for an enzymatic activity.
 7. The method according to anyone of claims 1 to 6, characterised in that the assembly of amino acidsequences has at least one substrate for each of at least two differentenzymatic activities.
 8. The method according to any one of claims 1 to7, characterised in that the enzymatic activity is selected from thegroup comprising oxidoreductases, transferases, hydrolases, lyases andligases, and especially is selected from the group comprising kinases,sulphotransferases, glycosyl transferases, acetyl transferases, farnesyltransferases, palmytyl transferases, phosphatases, sulphatases,esterases, lipases, acetylases and proteases.
 9. The method according toany one of claims 1 to 8, characterised in that the detection of areaction between the amino acid sequences immobilised on the assemblyand the enzymatic activity is repeated many times, preferably atintervals of time.
 10. The method according to any one of claims 1 to 9,characterised in that the enzymatic activity is determined in a sampleand the sample is preferably selected from the group comprising urine,liquor, sputum, stool, lymph fluid, other body fluids, cell lysates,tissue lysates, organ lysates, extracts, raw extracts, purifiedpreparations and unpurified preparations.
 11. The method according toany one of claims 1 to 10, characterised in that the surface is anon-porous surface.
 12. The method according to any one of claims 1 to11, characterised in that the support material is glass.
 13. The methodaccording to any one of claims 1 to 12, characterised in that the aminoacid sequence is immobilised via a sulphur-comprising group on thesurface.
 14. An assembly of a plurality of amino acid sequences on asurface wherein the amino acid sequences are directionally immobilisedon the planar surface of a support material, characterised in that atleast one of the amino acid sequences is a substrate for an enzymaticactivity, wherein a change in the molecular weight takes place on thesubstrate as a result of the enzymatic activity.
 15. The assemblyaccording to claim 14, characterised in that the change in the molecularweight takes place as a result of the formation or cleaving of acovalent bond on the substrate.
 16. The assembly according to claim 14or claim 15, characterised in that the assembly of amino acid sequenceshas at least one substrate for each of at least two different enzymaticactivities.
 17. The assembly according to any one of claims 14 to 16,characterised in that the planar surface is a non-porous surface. 18.The assembly according to claims 14 to 17, characterised in that thesupport material is selected from the group comprising silicates,ceramic, glass, metals and organic support materials.
 19. The assemblyaccording to any one of claims 14 to 18, characterised in that the aminoacid sequences are selected from the group comprising peptides,oligopeptides, polypeptides and proteins as well as their respectivederivatives.
 20. The assembly according to any one of claims 14 to 19,characterised in that each amino acid sequence or group of amino acidsequences has a defined arrangement relative to another amino acidsequence or groups of amino acid sequences.
 21. A support comprising anassembly according to any one of the preceding claims.
 22. The supportaccording to claim 21, characterised in that the support comprises abase support material.
 23. The support according to claim 21 or 22,characterised in that the assembly of a plurality of amino acidsequences is arranged on one or a plurality of surfaces of the support.24. The support assembly comprising at least two supports according toany one of claims 21 to 23, wherein respectively two supports areseparated by a gap.
 25. The support assembly according to claim 24,characterised in that at least one assembly on a first support is facingat least one assembly on a second support.
 26. The support assemblyaccording to claim 24 or claim 25, characterised in that the gap has awidth of around 0.01 mm to 10 mm, preferably around 0.1 mm to 2 mm, andmore preferably around 0.5 mm to 1 mm.
 27. The use of an assemblyaccording to any one of claims 14 to 20 and/or a support according toany one of claims 21 to 23 and/or a support assembly according to anyone of claims 24 to 26 in a method according to any one of claims 1 to13.